Algorithms for sequence determinations

ABSTRACT

The invention provides methods of determining a consensus sequence from multiple raw sequencing reads of a nucleic acid target. The nucleic acid target includes an anchor segment of known sequence and an adjacent segment of unknown sequence. The anchor segment provides a means to assess the quality of a raw target sequencing read. Raw target sequencing reads meeting or exceeding a threshold are assigned to an accepted class. The consensus sequence of the adjacent segment can be determined from raw target sequencing reads in the accepted class. Successive polling steps determine successive consensus nucleobases in a nascent sequence of the adjacent segment. Raw target sequencing reads can be removed or reintroduced from the accepted class depending on their correspondence to the most recently determined consensus nucleobase and/or the nascent sequence.

CROSS-REFERENCE TO RELATED APPLICATION

The present applications is a nonprovisional and claims the benefit of 61/539,440 filed Sep. 26, 2011 incorporated by reference in its entirety for all purposes.

BACKGROUND

Over the past decade, DNA sequencing throughput has increased over 50-fold. Advances in DNA sequencing have revolutionized the fields of cellular and molecular biology. High-throughput sequencing platforms include the 454 FLX™ or 454 TITANIUM™ (Roche), the SOLEXA™ Genome Analyzer (Illumina), the HELISCOPE™ Single Molecule Sequencer (Helicos Biosciences), the SOLID™ DNA Sequencer (Life Technologies/Applied Biosystems) instruments), SMRT™ technology developed by Pacific Biosystems, as well as other platforms still under development by companies such as Intelligent Biosystems.

Although such sequencing platforms generate vast amounts of sequencing data including multiple reads of the same target sequence, difficulties remain in deducing correct sequences present in a sample due to errors introduced by the high-throughput sequencing methods. With the high error rate, it is difficult to identify the majority species consistently and reliably. It is even more difficult to identify the minority species that differ little from the majority species and to determine their prevalence. Most sequence alignment-based methods alone cannot overcome high frequencies of error.

SUMMARY OF THE CLAIMED INVENTION

The invention provides computer-implemented methods of developing a consensus sequence from a plurality of sequencing reads of a nucleic acid target. Such methods involve (a) receiving a population of raw target sequencing reads of a nucleic acid target comprising an anchor segment and an adjacent segment; the anchor segment being of known sequence and the adjacent segment being of unknown sequence; and at least some of the raw target sequencing reads containing sequencing errors; (b) evaluating the accuracy of sequencing of the anchor segment in different raw target sequencing reads by comparing raw target sequencing reads of the anchor segment with the known sequence of the anchor segment; (c) assigning a subset of the raw target sequencing reads into an accepted class based on reaching at least a threshold level of accuracy of the sequencing of the anchor segment; (d) polling nucleobases at a position equidistant to the anchor segment sequence in raw target sequencing reads in the accepted class to determine a consensus nucleobase, which consensus nucleobase is assigned as the first nucleobase of a nascent sequence of the adjacent segment; (e) assigning raw target sequencing reads having the consensus nucleobase determined in the prior polling step to remain in an accepted class and assigning raw target sequencing reads lacking the consensus nucleobase determined in the prior polling step to the rejected class; (f) optionally reassigning a raw target sequencing read from the rejected class to the accepted class by scoring similarity of the raw target sequencing read to the nascent sequence and reintroducing the raw target sequencing read if the sequence similarity reaches at least a threshold level of similarity; and (g) repeating steps (d), (e) and optionally (f), except that a repetition polls a position adjacent the position poled in the previous polling step for raw target sequencing reads having the consensus nucleobase polled in the previous step or in the case of a raw target sequencing read reassigned from the rejected class to the accepted class and not polled in the previous polling step or if polled not having the consensus nucleobase in the previous polling step, the polling polls a position adjacent the position aligned with the last nucleobase of the nascent sequence to determine a consensus nucleobase, and the consensus nucleobases determined in successive repetitions are assigned as successive nucleobases in the nascent sequence of the adjacent segment.

In some methods, step (f) is performed at least once. In some methods, step (g) is performed at least 20 times and step (f) at least 5 times. In some methods, step (g) is performed at least 100 times and step (f) at least 20 times. In some methods, the threshold for step (f) is at least 80% identity between the raw target sequencing read and nascent sequence when maximally aligned and a match between the last assigned nucleobase of the nascent sequence and corresponding nucleobase of the raw targeting sequencing read. In some methods, the threshold level of accuracy of the sequencing the anchor segment is based on percentage of sequence identity and/or location of matched nucleobases between a raw target sequencing read and the known anchor segment. In some methods the threshold level of accuracy requires a raw target sequencing read to have the correct nucleobase corresponding to the nucleobase of the anchor segment immediately adjacent the adjacent segment. In some methods, the nucleic acid target includes a nucleobase variation at a position and when step (g) polls the position it determines two consensus nucleobases for the position, wherein the nascent sequence is branched into two nascent sequences differing between the two consensus nucleobases and the consensus nucleobase determined in further repetitions of step (g) is assigned to both nascent sequences. In some methods, the nucleic acid target comprises first and second anchor segments at opposing ends of the nucleic acid target and the raw target sequencing reads include a first group of raw target sequencing reads of the first anchor segment and an adjacent segment and a second group of raw target sequencing reads of the second anchor segment and an adjacent segment; the first and second groups being raw sequencing reads of opposing strands of the nucleic acid target and the method is performed on the first and second groups of raw target sequencing reads to determine consensus sequences of opposing strands of the target nucleic acid.

In some methods, the raw sequencing reads comprise raw sequencing reads of first and second nucleic acid targets, the first nucleic acid target comprising the anchor segment linked to a first adjacent segment and the second nucleic acid target comprising the anchor segment linked to a second adjacent segment. In some methods, the first and second adjacent segments are overlapping segments. In some methods, the first and second adjacent segments are fragments of the same contiguous polynucleotide. In some methods, the first and second adjacent segments are nonoverlapping segments. In some methods, the raw sequencing reads comprising raw sequence reads of a plurality of nucleic acid targets, the different nucleic acid targets comprising the anchor segment linked to different adjacent segments; the different adjacent segments including overlapping and nonoverlapping segments. In some methods, a strand of the anchor segment is a primer or primer binding site incorporated into the nucleic acid target. In some methods, a strand of the anchor segment has 4-120 nucleobases, or 8-30 nucleobases. In some methods, the anchor segment is an oligonucleotide ligated to a nucleic acid fragment to be sequenced. In some methods, the anchor segment and adjacent segment are contiguous segments in a nucleic acid from nature. In some methods, the anchor segment is a repeat sequence.

Some methods also involve outputting the sequence of at least part of the adjacent segment. Some methods also involve synthesizing a nucleic acid sequence having a sequence comprising at least part of the adjacent segment. Some methods also include experimentally determining the population of raw target sequencing reads of the target nucleic acid.

In some methods, the population of raw target sequencing reads is determined by a sequencing-by-synthesis method. In some methods, the sequencing method is single-molecule sequencing. In some methods, the sequencing method is single-molecule real time sequencing. In some methods, the nucleic acid target is in the form of a circular template. In some methods, the nucleic acid target is a homogeneous population of the same nucleic acid molecule.

In some methods the nucleic acid target is a heterogeneous population of variant nucleic acid molecules. In some methods, the variant nucleic acid molecules are variant nucleic acid molecules of the same virus. In some method, the virus is HIV or HCV. In some methods, the variants are allelic variants. In some methods, the nucleic acid target is a circular DNA molecule and the raw target sequencing reads comprise alternating reads of the anchor segment and the adjacent segment. In some methods, the reads of the adjacent segment comprise reads of alternating strands of the adjacent segment. In some methods, the circular DNA molecule is formed by ligating first and second hairpin anchor segments to the adjacent segment. In some methods, the first and second hairpin anchor segments are the same. In some methods, the first and second hairpin anchors are different and the raw target sequencing reads comprise alternating reads of the first and second hairpin anchor segments.

Some methods also involve designating a segment of the nascent sequence of the adjacent segment as a new anchor segment and repeating the method to determine a consensus sequence of an adjacent segment adjacent the new anchor segment.

The invention further provides a computer program product for analyzing a nucleic acid target, comprising (a) code for receiving a population of raw target sequencing reads of a nucleic acid target comprising an anchor segment and an adjacent segment; the anchor segment being of known sequence and the adjacent segment being of unknown sequence; and at least some of the raw target sequencing reads containing sequencing errors; (b) code for evaluating the accuracy of sequencing of the anchor segment in different raw target sequencing reads by comparing the raw target sequencing reads of the anchor segment with the known sequence of the anchor segment; (c) code for assigning a subset of the raw target sequencing reads into an accepted class based on reaching at least a threshold level of accuracy of the sequencing of the anchor segment; (d) code for polling nucleobases at a position equidistant to the anchor segment sequence in raw target sequencing reads in the accepted class to determine a consensus nucleobase, which consensus nucleobase is assigned as the first nucleobase of a nascent sequence of the adjacent segment; (e) code for assigning raw target sequencing reads having the consensus nucleobase determined in the prior polling step to remain in an accepted class and assigning raw target sequencing reads lacking the consensus nucleobase determined in the prior polling step to the rejected class; (f) code for optionally reassigning a raw target sequencing read from the rejected class to the accepted class by scoring similarity of the raw target sequencing read to the nascent sequence and reintroducing the raw target sequencing read if the sequence similarity reaches at least a threshold level of similarity; and (g) code for repeating steps coded in (d), (e) and optionally (f), except that a repetition polls a position adjacent the position poled in the previous polling step for raw target sequencing reads having the consensus nucleobase polled in the previous step or in the case of a raw target sequencing read reassigned from the rejected class to the accepted class and not polled in the previous polling step or if polled not having the consensus nucleobase in the previous polling step, the polling polls a position adjacent the position aligned with the last nucleobase of the nascent sequence to determine a consensus nucleobase, and the consensus nucleobases determined in successive repetitions are assigned as successive nucleobases in the nascent sequence of the adjacent segment.

In some computer program products, the threshold in (f) is at least 80% identity between the raw target sequencing read and nascent sequence when maximally aligned and a match between the last assigned nucleobase of the nascent sequence and corresponding nucleobase of the raw targeting sequencing read. In some computer program products, the threshold level of accuracy of the sequencing the anchor segment is based on percentage of sequence identity and/or location of matched nucleobases between a raw target sequencing read and the known anchor segment. In some computer program products, the threshold level of accuracy requires a raw target sequencing read to have the correct nucleobase corresponding to the nucleobase of the anchor segment immediately adjacent the adjacent segment. In some computer program products, the raw sequencing reads comprise raw sequencing reads of first and second nucleic acid targets, the first nucleic acid target comprising the anchor segment linked to a first adjacent segment and the second nucleic acid target comprising the anchor segment linked to a second adjacent segment. In some computer program products, the first and second adjacent segments are overlapping segments. In some computer program products, the first and second adjacent segments are fragments of the same contiguous polynucleotide. In some computer program products, the first and second adjacent segments are nonoverlapping segments. In some computer program products, the raw sequencing reads comprise raw sequencing reads of a plurality of nucleic acid targets, the different nucleic acid targets comprising the anchor segment linked to different adjacent segments; the different adjacent segments including overlapping and nonoverlapping segments. In some computer program products, the strand of the anchor segment is a primer incorporated into the nucleic acid target. In some computer program products, the strand of the anchor segment has 4-120 nucleobases, or 8-30 nucleobases. In some computer program products, the anchor segment is an oligonucleotide ligated to a nucleic acid fragment to be sequenced. In some computer program products, the anchor segment and adjacent segment are contiguous segments in a nucleic acid from nature. In some computer program products, the anchor segment is a repeat sequence. Some computer program products further comprise code for outputting the sequence of at least part of the adjacent segment. In some computer program products, the nucleic acid target is a homogeneous population of the same nucleic acid molecule. In some computer program products, the nucleic acid target is a heterogeneous population of variant nucleic acid molecules. In some computer program products, the variant nucleic acid molecules are variant nucleic acid molecules of the same virus. In some computer program products, the virus is HIV or HCV. In some computer program products, the variants are allelic variants.

The invention further provides a system for analyzing a nucleic acid target, comprising: (1) a system bus; (2) a memory coupled to the system bus; and (3) a processor coupled to the system bus operatively disposed to: (a) receive a population of raw target sequencing reads of a nucleic acid target comprising an anchor segment and an adjacent segment; the anchor segment being of known sequence and the adjacent segment being of unknown sequence; and at least some of the raw target sequencing reads containing sequencing errors; (b) evaluate the accuracy of sequencing of the anchor segment in different raw target sequencing reads by comparing raw target sequencing reads of the anchor segment with the known sequence of the anchor segment; (c) assign a subset of the raw target sequencing reads into an accepted class based on reaching at least a threshold level of accuracy of the sequencing of the anchor segment; (d) poll nucleobases at a position equidistant to the anchor segment sequence in raw target sequencing reads in the accepted class to determine a consensus nucleobase, which consensus nucleobase is assigned as the first nucleobase of a nascent sequence of the adjacent segment; (e) assign raw target sequencing reads having the consensus nucleobase determined in the prior polling step to remain in an accepted class and assigning raw target sequencing reads lacking the consensus nucleobase determined in the prior polling step to the rejected class; (f) optionally reassign a raw target sequencing read from the rejected class to the accepted class by scoring similarity of the raw target sequencing read to the nascent sequence and reintroducing the raw target sequencing read if the sequence similarity reaches at least a threshold level of similarity; and (g) repeat steps (d), (e) and optionally (f), except that a repetition polls a position adjacent the position poled in the previous polling step for raw target sequencing reads having the consensus nucleobase polled in the previous step or in the case of a raw target sequencing read reassigned from the rejected class to the accepted class and not polled in the previous polling step or if polled not having the consensus nucleobase in the previous polling step, the polling polls a position adjacent the position aligned with the last nucleobase of the nascent sequence to determine a consensus nucleobase, and the consensus nucleobases determined in successive repetitions are assigned as successive nucleobases in the nascent sequence of the adjacent segment.

In some systems, the threshold in (f) is at least 80% identity between the raw target sequencing read and nascent sequence when maximally aligned and a match between the last assigned nucleobase of the nascent sequence and corresponding nucleobase of the raw targeting sequencing read. In some systems, the threshold level of accuracy of sequencing the anchor segment is based on percentage of sequence identity and/or location of matched nucleobases between a raw target sequencing read and the known anchor segment. In some systems, the threshold level of accuracy requires a raw target sequencing read to have the correct nucleobase corresponding to the nucleobase of the anchor segment immediately adjacent the adjacent segment. In some systems, the raw sequencing reads comprise raw sequencing reads of first and second nucleic acid targets, the first nucleic acid target comprising the anchor segment linked to a first adjacent segment and the second nucleic acid target comprising the anchor segment linked to a second adjacent segment. In some systems, the first and second adjacent segments are overlapping segments. In some systems, the first and second adjacent segments are fragments of the same contiguous polynucleotide. In some systems, the first and second adjacent segments are nonoverlapping segments. In some systems, the raw sequencing reads comprising raw sequence reads of a plurality of nucleic acid targets, the different nucleic acid targets comprising the anchor segment linked to different adjacent segments; the different adjacent segments including overlapping and nonoverlapping segments. In some systems, the strand of the anchor segment is a primer incorporated into the nucleic acid target. In some systems, the strand of the anchor segment has 4-120 nucleobases. In some systems, the strand of the anchor segment has 8-30 nucleobases. In some systems, the anchor segment is an oligonucleotide ligated to a nucleic acid fragment to be sequenced. In some systems, the anchor segment and adjacent segment are contiguous segments in a nucleic acid from nature. In some systems, the anchor segment is a repeat sequence. In some systems, the processor is operatively disposed to outputting the sequence of at least part of the adjacent segment. In some systems, the nucleic acid target is a homogeneous population of the same nucleic acid molecule. In some systems, the nucleic acid target is a heterogeneous population of variant nucleic acid molecules. In some systems, the variant nucleic acid molecules are variant nucleic acid molecules of the same virus. In some systems, the virus is HIV or HCV. In some systems, the variants are allelic variants.

The invention further provides methods of differentially treating a patient population. Such methods involve sequencing samples from members of the patient population; wherein for each sample the sequencing comprises: (a) receiving a population of raw target sequencing reads of a nucleic acid target comprising an anchor segment and an adjacent segment; the anchor segment being of known sequence and the adjacent segment being of unknown sequence; and at least some of the raw target sequencing reads containing sequencing errors; (b) evaluating the accuracy of sequencing of the anchor segment in different raw target sequencing reads by comparing raw target sequencing read of the anchor segment with the known sequence of the anchor segment; (c) assigning a subset of the raw target sequencing reads into an accepted class based on reaching at least a threshold level of accuracy of the sequencing of the anchor segment; (d) polling nucleobases at a position adjacent the anchor segment sequence in raw target sequencing reads in the accepted class to determine a consensus nucleobase, which consensus nucleobase is assigned as the first nucleobase of a nascent sequence of the adjacent segment; (e) assigning raw target sequencing reads having the consensus nucleobase determined in the prior polling step to remain in an accepted class and assigning raw target sequencing reads lacking the consensus nucleobase determined in the prior polling step to the rejected class; (f) optionally reassigning a raw target sequencing read from the rejected class to the accepted class by scoring similarity of the raw target sequencing read to the nascent sequence and reintroducing the raw target sequencing read if the sequence similarity reaches at least a threshold level of similarity; and (g) repeating steps (d), (e) and optionally (f), except that a repetition polls a position adjacent the position poled in the previous polling step for raw target sequencing reads having the consensus nucleobase polled in the previous step or in the case of a raw target sequencing read reassigned from the rejected class to the accepted class and not polled in the previous polling step or if polled not having the consensus nucleobase in the previous polling step, the polling polls a position adjacent the position aligned with the last nucleobase of the nascent sequence to determine a consensus nucleobase, and the consensus nucleobases determined in successive repetitions are assigned as successive nucleobases in the nascent sequence of the adjacent segment. Different members of the patient population receive different treatment regimes depending on the determined sequence for the sample from each member.

The invention further provides computer-implemented methods of analyzing a nucleic acid target. Such methods involve (a) receiving a population of raw target sequencing reads of a nucleic acid target comprising an anchor segment and an adjacent segment; the anchor segment being of known sequence and the adjacent segment being of unknown sequence; and at least some of the raw target sequencing reads containing sequencing errors; (b) evaluating the accuracy of sequencing of the anchor segment in different raw target sequencing reads by comparing raw target sequencing reads of the anchor segment with the known sequence of the anchor segment; (c) assigning a subset of the raw target sequencing reads into an accepted class based on the accuracy of sequencing of the anchor segment in the raw target sequencing reads; and (d) determining a sequence of the anchor segment from raw target sequencing reads in the accepted class.

In some methods, step (d) comprises polling nucleobases at corresponding positions in raw target sequencing reads in the accepted class to determine a consensus nucleobase, which consensus nucleobase is assigned as the first nucleobase of a nascent sequence of the adjacent segment and wherein step (d) is repeated and the consensus nucleobases determined in successive repetitions are assigned as successive nucleobases in the nascent sequence of the adjacent segment. Some methods also involve assigning raw target sequencing reads having the consensus nucleobase determined in the prior polling step to remain in an accepted class and assigning raw target sequencing reads lacking the consensus nucleobase determined in the prior polling step to a rejected class. Some methods also involve designating a segment of the sequence of the adjacent segment as a new anchor segment and repeating the method to determine a sequence of an adjacent segment adjacent the new anchor segment.

The invention further provides a computer program product for analyzing a nucleic acid target, comprising code for receiving a population of raw target sequencing reads of a nucleic acid target comprising an adapter segment and an adjacent segment; the adapter segment being of known correct sequence and the adjacent segment being of unknown sequence; and at least some of the raw target sequences containing sequencing errors; code for evaluating the accuracy of sequencing of the adapter segment in different raw target sequences by comparing raw target sequencing reads of the anchor segment with the known correct sequence of the adapter segment; code for assigning a subset of the raw target sequences into an accepted class based on the accuracy of sequencing of the adapter segment in the raw target sequences; code for aligning at least some of the raw target sequences from the accepted class; and code for determining a sequence of at least part of the adjacent segment from the aligned sequences.

The invention further provides a system for analyzing a nucleic acid target, comprising: (a) a system bus; (b) a memory coupled to the system bus; and (c) a processor coupled to the system bus for receiving a population of raw target sequencing reads of a nucleic acid target comprising an adapter segment and an adjacent segment; the adapter segment being of known correct sequence and the adjacent segment being of unknown sequence; and at least some of the raw target sequences containing sequencing errors; evaluating the accuracy of sequencing of the adapter segment in different raw target sequences by comparing the raw target sequencing reads of the anchor segment with the known correct sequence of the adapter segment; assigning a subset of the raw target sequences into an accepted class based on the accuracy of sequencing of the adapter segment in the raw target sequences; aligning at least some of the raw target sequences from the accepted class; and determining a sequence of at least part of the adjacent segment from the aligned sequences.

The invention further provides methods of differentially treating a patient population. Such methods involve sequencing samples from members of the patient population; wherein for each sample the sequencing comprises receiving a population of raw target sequencing reads of a nucleic acid target comprising an adapter segment and an adjacent segment; the adapter segment being of known correct sequence and the adjacent segment being of unknown sequence; and at least some of the raw target sequences containing sequencing errors; evaluating the accuracy of sequencing of the adapter segment in different raw target sequences by comparing raw target sequencing reads of the anchor segment with the known correct sequence of the adapter segment; assigning a subset of the raw target sequences into an accepted class based on the accuracy of sequencing of the adapter segment in the raw target sequences; aligning at least some of the raw target sequences from the accepted class; and determining a sequence of at least part of the adjacent segment from the aligned sequences; wherein different members of the patient population receive different treatment regimes depending on the determined sequence for the sample from each member.

DEFINITIONS

Brief descriptions of some of the terms used in this application appear below. Some of these terms are further described in the rest of the specification.

A nucleobase is the base component of a nucleotide including any of the natural bases adenine (A), cytosine (C), guanine (G) and thymine (T) (for DNA) and A, C, G, and uracil (U) (for RNA) or analogs thereof subjectable to a sequencing reaction (e.g., support template-dependent incorporation of a complementary nucleobase). Nucleobases are sometimes referred to simply as bases.

A nucleobase attached to a sugar, can be referred to as a nucleobase unit, or monomer. Sugar moieties of a nucleic acid can be ribose, deoxyribose, dideoxyribose or similar compounds, e.g., with 2′ methoxy or 2′ halide substitutions. Nucleotides and nucleosides are examples of nucleobase units.

A nucleic acid target is the nucleic acid unit that is the subject of a sequencing reaction and gives rise to a sequencing read. A nucleic acid target comprises an anchor segment of known sequence and an adjacent sequence whose sequence is to be determined.

A raw target sequencing read is a contiguous sequence of nucleobases assigned during a sequencing reaction on a nucleic acid target. A raw target sequencing read may contain sequencing error(s). Thus raw target sequencing reads of the same nucleic acid target can differ from one another by virtue of sequencing errors.

Raw target sequencing reads can be assigned into an accepted class or rejected class. Raw target sequencing reads in the accepted class have passed a quality control measure. The quality control measure can be that the accuracy of sequencing of the anchor segment at least reaches a defined threshold, or that a raw target sequencing read contains a consensus nucleotide at an immediately previously polled position or a raw target sequencing read exceeds a threshold level of sequence similarity with the nascent sequence. Conversely raw target sequencing reads in the rejected class have failed a quality control measure. Typically, this quality control measure is failure to contain a consensus nucleobase at a polled position. Raw target sequencing can be assigned from the accepted class to the rejected class and vice versa as described below.

Polling compares the nucleobase occupying corresponding positions among raw target sequencing reads to determine a consensus nucleobase for that position.

A nascent sequence refers to a string of contiguous nucleobases identified by repeated polling cycles. The nascent sequence is the sequence of at least part of the adjacent segment of a nucleic acid target.

A threshold relates to one or more criteria for evaluating a nucleotide sequence, such as a raw target sequencing read. Such a threshold can be stored as code or provided by user input, or selected from a menu of possible thresholds when the method is performed.

In pairwise comparisons between two nucleic acid sequences, the nucleic acids are maximally aligned when the number of nucleobase matches is greatest. Percentage sequence identity can be defined as the number of matched nucleobases between aligned sequences divided by the number of nucleobases in one of the sequences (usually the known sequence if one sequence is known and the other is not). Extra nucleobases in an unknown sequence flanking the part of the unknown aligned with a known sequence are not scored.

Some or all of a raw target sequence read corresponds with the nucleic acid target (i.e., has the same sequence as a strand of the nucleic acid target other than sequence errors). The portion of a raw target sequencing read corresponding to an anchor segment of a nucleic acid target is the portion of the raw target sequencing aligned with the known sequence of the anchor segment when the raw target sequencing read is maximally aligned with the anchor segment. A portion of the raw targeting sequencing read adjacent to the segment corresponding to the anchor segment corresponds with the adjacent segment of the nucleic acid target.

A corresponding position in two or more nucleic acid sequences is a position aligned between the sequences when the sequences are maximally aligned over their entire length or at least a defined window thereof including the corresponding position (e.g., at least 10 or 20 nucleotides).

A “primer” is an oligonucleotide, typically between about 10 to 100 nucleotides in length, capable of selectively binding to a specified nucleic acid or “template” by hybridizing with the template. The primer provides a point of initiation for polymerase-mediated template-directed synthesis of a nucleic acid complementary to the template. Primers hybridizing to opposing strands of a double-stranded sequence are referred to as forward and reverse primers. An oligonucleotide primer used to initiate a sequencing reaction is referred to as a sequencing primer.

A “sequence variation” refers to a point or region of variation between two related nucleic acid molecules (e.g., at least 50% sequence identity and usually, at least 75%, 90, 95 or 99% sequence identity). A variation can be an insertion, deletion or substitution of one or more nucleobase differences between two nucleic acid molecules. A variation can be natural, such as allelic, or between species, strains or isolates or induced. Variations can be between different molecules of viral nucleic acids in a sample. Variations can be germline or somatic. A variation in nucleotide sequence can have no effect on an encoded amino acid sequence due to degeneracy of the code or can result in a corresponding amino acid change. If there is an amino acid change, the change may or may not affect the function of the encoded protein. If the change is to a stop codon, the encoded protein becomes prematurely truncated.

A copy of an anchor segment or adjacent segment or read thereof means an identical copy or substantially identical copy (e.g., at least 80% sequence identity) differing as a result of nucleobase unit misincorporations in template-dependent extension or sequencing errors.

Description of a range by integers representing the boundaries of the range also refers to all subranges defined by integers within the range.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a sequence determination algorithm.

FIG. 2 shows a configuration of a device for analyzing a nucleic acid target.

FIGS. 3A-F show sequencing using hairpin anchor segments.

FIGS. 4A-D show determining a consensus sequence by nucleobase polling.

DETAILED DESCRIPTION I. General

The invention provides methods of determining a consensus sequence from multiple raw sequencing reads of a nucleic acid target. The nucleic acid target includes an anchor segment of known sequence and an adjacent segment of unknown sequence. The anchor segment provides a means to assess the quality of a raw target sequencing read. Because the anchor segment is of known sequence, comparison of the portion of the raw target sequencing read corresponding to the anchor segment provides a measure of quality of the raw target sequencing read. Raw target sequence reads exceeding a threshold level of accuracy are used in determining a consensus sequence for the adjacent target sequence. Raw target sequencing reads not meeting the threshold level of accuracy can be excluded from subsequent analysis.

The consensus sequence of the adjacent segment can be determined from raw target sequencing reads passing the threshold test by polling the target sequence reads at a corresponding position starting with a position adjacent the anchor segment. Successive polling steps can determine successive consensus nucleobases in a nascent sequence of the adjacent segment. Raw target sequencing reads can be removed or reintroduced from the accepted class depending on their correspondence to the most recently determined consensus nucleobase and/or the nascent sequence.

II. Nucleic Acid Target and Sequencing Read Thereof

The nucleic acid unit that is subject of a sequencing reaction and gives rise to a sequencing read (or two sequencing reads from opposing strands) is referred to as a nucleic acid target. A nucleic acid target can be single- or double-stranded, RNA or DNA. A nucleic acid target can be linear or circular. A nucleic acid target includes an anchor segment of known sequence and an adjacent segment whose sequence is to be determined. The adjacent segment can have a single unique sequence, can be a simple mixture of two variants (e.g., bi-allelic variants) or a complex mixture of variant sequences (e.g., a particular mRNA from a viral sample in which multiple viral strains are represented). The nucleic acid target can be of any length but preferably less than 200%, more preferably from 50% to 200%, of the maximum length of raw target sequencing read obtainable with whatever methodology is used. For example, the length of the nucleic acid target is sometimes from 20-50,000 nucleobases or by for a double stranded nucleic acid target. The nucleic acid target or its adjacent segment can be part of a larger target molecule, such as a gene, viral genome, chromosome or full genome. In this case, as in other sequencing methods, the larger target molecule can be broken down into fragments each of which can constitute a nucleic acid target or an adjacent segment of a nucleic acid target for purposes of the present methods. The sequences of multiple nucleic acid targets or adjacent segments thereof can be compiled from overlaps to provide the sequence of a larger nucleic acid molecule.

The anchor segment refers to a segment of known sequence present in the nucleic acid target. Anchors can be or various lengths, e.g., 8-30, 4-10, 4-20, 4-30, 4-50, or 4-120 nucleobases or base pairs). Anchor segments can be formed from deoxyribonucleotides or ribonucleotides or in some cases nucleotide analogs that can be subject of sequencing reactions.

Anchor segments can be nucleic acid sequences that are heterologous (i.e., not naturally associated with adjacent segment). Examples of heterologous anchor segments include primers or portions thereof used in amplifying adjacent segments, binding sites for sequencing primers, oligonucleotides ligated or otherwise attached to adjacent segments, such as SMRT Bell™ hairpin structures. Such ligated oligonucleotides including SMRT Bell structures can also serve as primer binding sites. Anchor segments can also be nucleic acid sequences naturally associated with the adjacent nucleic acid segment. Examples of anchor segments that are endogenous to the nucleic acid template include portions of a gene of known sequence, regulatory sequences, and repetitive sequences. The anchor segment preferably has a single (i.e., without sequence variation) completely known sequence. However, anchor segments which are of substantially completely known sequences, i.e., at least 80, 95, or 99% of nucleobases are known and without nucleobase variation can also be used.

Many sequencing methods already incorporate a segment that can serve as an anchor segment in the course of preparing a sequence template. In SMRT™ technology, a nucleic acid to be sequenced is ligated to hairpin structures (the same or different from each other), which can serve as anchor segments, one anchor segment joining at each end, forming circular template. The circular template includes strands of the nucleic acid to be sequenced (adjacent segment) and the hairpin anchor segments. Such a circular template can be sequenced in a single well to generate a sequencing read including alternating target strand and anchor segments (e.g., anchor segment 1, first strand of adjacent segment, anchor segment 2, second strand of target segment, anchor segment 1, first strand of adjacent segment, anchor segment 2, second strand of target segment and so forth) Oligonucleotide anchors can be ligated to libraries of nucleic acids to be sequenced (Illumina, Inc., 454 Corporation, SOLiD). Primers for the extension of a polynucleotide complementary to a nucleic acid to be sequenced e.g., poly (T) oligonucleotides, can also be used as anchors. Target nucleic acids can contain one, two or more copies of an anchor segment, each copy interspersed between copies of the adjacent segment (and/or its complement).

Performing a sequencing reaction on a nucleic acid target gives rise to a population of raw target sequencing reads of the nucleic acid target. A raw target sequencing read includes sequence of both the adapter segment and adjacent segment. The length of raw target sequencing of the same target can show some variation. A raw target sequencing read of an anchor segment and an adjacent segment can include the complete anchor segment or a designated portion of at least 10, 15, 20 or 30 nucleotides thereof abutting an adjacent sequence, and at least some, and preferably at least 25, 50, 75, 95 or 100% of the adjacent segment. A raw target sequence read refers to a contiguous nucleobase sequence assigned during a sequencing reaction performed on nucleic acid target. If the nucleic acid is double-stranded, the raw target sequencing read can correspond to either strand of the nucleic acid target. If the nucleic acid target is single-stranded, the raw target sequencing read can correspond to the nucleic acid target strand or its complement. The sequencing reaction can be performed using any type of sequencing methodology. Depending on the type of sequencing methodology used, the reaction provides a series of signals that are individually interpreted to mean one of A, C, T, G or U (or analogs thereof). This initial assignment of contiguous nucleobases forming the raw target sequence read may contain one or more errors (i.e., insertions, deletions, substitutions and combinations thereof). Different raw target sequences of the same nucleic acid target typically contain errors in different positions. Errors can result from misincorporation of nucleotides in amplification or sequencing, reading errors associated with instrumentation and the enzymatic sequencing process, and errors introduced in base-calling.

Raw target sequencing reads can be in the form first generated by a sequencing reaction without any processing to remove errors or can have been subject to partial processing to remove some errors but in which some sequencing errors remain.

A population of raw target sequencing reads of a nucleic acid target can be generated by repeatedly sequencing the same nucleic acid target molecule, sequencing a nucleic acid containing multiple copies of the nucleic acid target (e.g., repeats generated by rolling circle replication), or by sequencing multiple individual copies of the nucleic acid target or larger molecule containing some or all of the nucleic acid target. Examples of methods of generating replicate sequence information from a single molecule are provided, e.g., in U.S. Pat. No. 7,476,503; US 2009/0298075, 2010/0075309, 2010/0075327, 2010/0081143. For example, a circular template can be used to generate replicate sequence reads of the target sequence by allowing a polymerase to synthesize a linear concatemer by continuously generating a nascent strand from multiple passes around the template molecule. The nascent strand can contain alternating reads of an anchor segment and adjacent segment. Optionally, the anchor segment itself alternates between first and second anchor segment and the adjacent segment alternates between its two strands. The population of raw target sequencing reads of a nucleic acid target may or may not begin and end at the same position as each other. However, a nucleic acid read should include at least sufficient numbers of nucleobases of the anchor segment to permit evaluation of accuracy of sequencing of the anchor segment, and at least some of the adjacent segment. Preferably raw target sequencing reads of the same target nucleic acid begin at the same point, include all of an anchor segment and as much of an adjacent segment as is compatible with the sequencing technology. Often there is some variation in the read length of different raw sequencing reads, which is preferably reflected in variation of the length of adjacent segment included in the read. The read lengths of different sequencing technology vary widely. Thus, the amount of adjacent sequence included in a read length can vary from e.g., 10 nucleobases to 50,000 nucleobases.

Raw target sequencing reads may or may not be provided with additional information as well as pure sequence data. Additional information can include estimations of per-position accuracy, features of underlying sequencing technology output (e.g., trace characteristics, integrated counts per peak, shape/height/width of peaks, distance to neighboring peaks), signal-to-noise ratios, power-to-noise ratio, signal strength, and the like.

III. Generation of a Population of Raw Target Sequencing Reads of a Nucleic Acid Target (a) Template Preparation

Nucleic acid targets can be amplified before sequencing, or not amplified and used directly in sequencing. Amplification can be performed with a pair of forward and reverse primers as in conventional PCR. Optionally the forward and reverse primers include 5′ tails lacking complementary to the nucleic acid being amplified. Such tails can serve to provide a binding site for sequencing primers. Some or all of the forward and/or reverse primer can be used as the anchor segment of the nucleic acid target. The forward and reverse primers can serve as anchor segments for the opposing strands of a double-stranded nucleic acid target.

In other methods, after fragmenting a nucleic acid and repairing fragment ends, if needed, hairpin anchors can be ligated onto the ends of these fragments therefore forming a circularized template for sequencing. FIG. 3A shows a sample preparation using two hairpin anchors termed anchors I and II.

A DNA polymerase can be used to open the anchor-ligated fragments into circularized templates. The anchors serve as binding sites for sequencing primer(s) as well as their role in assessing quality of sequencing reads in the present methods. FIG. 3B shows generation of continuous reads of both strands of a fragment interspersed with anchors I and II. The reads, then, include multiple reads of the same fragment by the polymerase reading around the circular template multiple times. In FIG. 3B, four reads of forward strand and three reads of reverse strand have been generated before the sequencing reaction is terminated.

For analysis, the different sequencing reads can be segregated by an anchor segment. In this example, raw target sequences can be grouped into four subsets. In the first set, anchor I segments are aligned to provide a framework for base-polling sequences of forward strands adjacent to the 3′ end of the anchor I segment (FIG. 3C). In the second set, anchor II segments are aligned to provide a framework for base-polling sequences of reverse strands adjacent to the 3′ end of the anchor II (FIG. 3E). In the third and fourth sets, anchor I or II segments are aligned to provide frameworks for base-polling sequences of forward strands (FIG. 3F) and reverse strands (FIG. 3D) adjacent to the 5′ end of the anchor I and II. Anchors I and II can be the same or different from one another in terms of sequence.

Some amplification methods amplify many nucleic acid molecules in parallel. One such method is amplification on beads using emulsion PCR methods (see, e.g., US US2005/0042648, US2005/0079510, and US2005/0130173 and WO 05/010145). Another such method is amplification on a surface using bridge amplification to form nucleic acid clusters. Methods of generating nucleic acid clusters for use in high-throughput nucleic acid sequencing have been described (see, e.g., US No. 7,115,400, US 2005/0100900 and 2005/0059048, and WO 98/44151, WO 00/18957, WO 02/46456, WO 06/064199, and WO 07/010251. Bridge amplification refers to a solid phase replication method in which primers are bound to a solid phase, e.g., flow cell, microarray, and the like. The extension product from one bound primer forms a bridge to the other bound primer.

(b) Sequencing

One class of sequencing reactions that can be used are sequencing-by-synthesis (SBS) methods. Sequencing by synthesis refers to the sequencing of a nucleic acid sequence by synthesis of the complementary strand (see US 2007/0166705, 2006/0188901, 2006/0240439, 2005/0100900, 2006/0281109; U.S. Pat. No. 7,057,026; WO 05/065814, WO 06/064199 and WO 07/010251).

SBS techniques can utilize nucleotide monomers that have a label moiety or those that lack a label moiety. If a label is present, the monomers can have the same or different label as each other. If present, incorporation events can be detected based on a characteristic of the label(s), such as fluorescence of the label(s); a characteristic of the nucleotides such as molecular weight or charge; a byproduct of incorporation of the nucleotides, such as release of pyrophosphate or a hydrogen ion; or the like.

In some methods, the incorporation of nucleobase units is detected by measuring the release of a label from the nucleobase unit being incorporated. A preferred approach as with SMRTbell™ template sequence is to use nucleobase units fluorescently labeled on the terminal phosphate of the nucleobase unit. (Korlach et al., Nucleosides, Nucleotides and Nucleic Acids, 27:1072-1083, 2008. The label is cleaved from the nucleotide monomer on incorporation of the nucleotide into the polynucleotide. Accordingly, the label is not incorporated into a nascent nucleic acid, increasing the signal:background ratio.

Pyrosequencing detects the release of inorganic pyrophosphate (PPi) as particular nucleotides are incorporated into the nascent strand (Ronaghi, et al., Analytical Biochemistry 242(1):84-9, 1996; Ronaghi, M., Genome Res. 11(1):3-11, 2001; Ronaghi, et al., Science 281(5375):363, 1998; U.S. Pat. Nos. 6,210,891, 6,258,568 and 6,274,320). Released PPi can be detected by, e.g., a process in which the released PPi is immediately converted to adenosine triphosphate (ATP) by ATP sulfurylase, and the level of ATP generated is detected via luciferase-produced photons.

A hydrogen ion released on incorporation of a nucleotide can be detected as a change in voltage by for example the Ion Torrent machine (Life Technologies, Inc).

In another example, cycle sequencing is accomplished by stepwise addition of reversible terminator nucleotides containing, for example, a cleavable or photobleachable dye label. The technique was commercialized by Solexa (now Illumina Inc.), and described, for example, in U.S. Pat. Nos. 7,427,67, 7,414,163 and 7,057,026, and WO 91/06678 and WO 07/123744. The availability of fluorescently-labeled terminators in which both the termination can be reversed and the fluorescent label cleaved facilitates efficient cyclic reversible termination (CRT) sequencing. Polymerases can also be co-engineered to efficiently incorporate and extend from these modified nucleotides. In cases where two or more different nucleotides are present in a sequencing reagent, the different nucleotides can be distinguishable from each other. For example, the different nucleotides present in a sequencing reagent can have different labels and they can be distinguished using appropriate optics as exemplified by the sequencing methods developed by Solexa (now Illumina, Inc.).

SBS can utilize nucleotide monomers that have a terminator moiety or those that lack any terminator moieties. For SBS techniques that utilize nucleotide monomers having a terminator moiety, the terminator can be effectively irreversible under the sequencing conditions used as is the case for traditional Sanger sequencing which utilizes dideoxynucleotides, or the terminator can be reversible as is the case for sequencing methods developed by Solexa (now Illumina, Inc.). Methods utilizing nucleotide monomers lacking terminators include, for example, pyrosequencing and sequencing using γ-phosphate-labeled nucleotides. In methods using nucleotide monomers lacking terminators, the number of different nucleotides added in each cycle can be dependent upon the template sequence and the mode of nucleotide delivery. Reversible terminators/cleavable fluors can include fluor linked to the ribose moiety via a 3′ ester linkage (Metzker, Genome Res. 15:1767-1776 (2005). Other approaches have separated the terminator chemistry from the cleavage of the fluorescence label (Ruparel et al., Proc. Natl. Acad. Sci. USA 102: 5932-7 (2005). Ruparel et al. described the development of reversible terminators that used a small 3′ allyl group to block extension, but could easily be deblocked by a short treatment with a palladium catalyst. The fluorophore was attached to the nucleobase via a photocleavable linker that could easily be cleaved by a 30 second exposure to long wavelength UV light. Thus, either disulfide reduction or photocleavage can be used as a cleavable linker. Another approach to reversible termination is the use of natural termination that ensues after placement of a bulky dye on a dNTP. The presence of a charged bulky dye on the dNTP can act as an effective terminator through steric and/or electrostatic hindrance. The presence of one incorporation event prevents further incorporations unless the dye is removed. Cleavage of the dye removes the fluor and effectively reverses the termination (see U.S. Pat. Nos. 7,427,673 and 7,057,026).

Another class of sequencing reactions that can be used are nanopore sequencing methods. In nanopore sequencing, (Deamer, & Akeson, Trends Biotechnol. 18:147-151 (2000); Deamer & Branton, Acc. Chem. Res. 35:817-825 (2002); Li, et al., Nat. Mater. 2:611-615 (2003)), the target nucleic acid or nucleotides released from the target nucleic acid pass through a nanopore. The nanopore can be a synthetic pore or biological membrane protein, such as α-hemolysin. As the target nucleic acid or nucleotides pass through the nanopore, each base-pair (or base) can be identified by measuring fluctuations in the electrical conductance of the pore. (U.S. Pat. No. 7,001,792; Soni, G. V. & Meller, Clin. Chem. 53:1996-2001 (2007); Healy, K., Nanomed. 2:459-481 (2007); Cockroft, et al., J. Am. Chem. Soc. 130:818-820 (2008)).

Another class of sequencing reactions is sequencing by ligation (see U.S. Pat. No. 6,969,488, U.S. Pat. No. 6,172,218, and U.S. Pat. No. 6,306,597). A target nucleic acid is hybridized to an oligonucleotide and contacted with several probes and a ligase. Only a probe complementary to the target nucleic acid can be ligated to the oligonucleotide. The identity of the probe indicates part of the sequence of the target nucleic acid.

(c) Sequencing Platforms

Examples of sequencing platforms include the Genome Sequencer FLX System (Roche) that employs pyrosequencing to provide long read lengths and very high single-read accuracy, 454 FLX™ or 454 TITANIUM™ (Roche), the SOLEXA™ Genome Analyzer (Illumina), the HELISCOPE™ Single Molecule Sequencer (Helicos Biosciences), the SOLID™ DNA Sequencer (Life Technologies/Applied Biosystems) instruments) which performs sequencing by ligation, SMRT™ technology (Pacific Biosystems), Ion Torrent (LifeTech) as well as other platforms still under development by companies such as Intelligent Biosystems. Other sequencing platforms include OmniMoRA (Reveo, Inc. (Elmsford, N.Y.)), VisiGen® (VisiGen Biotechnologies, Inc. (Houston, Tex.), now Life Technologies (Carlsbad, Calif.)), SBS technology (Intelligent Bio-Systems (Waltham, Mass.)), or Hybridization-Assisted Nanopore Sequencing (HANS; NABsys Inc. (Providence, R.I.)), or the target fragment isolated may be sent to a third party for further analysis and/or sequencing (e.g., Really Tiny Stuff, Inc., Cohasset, Mass.).

A sequencing platform provided by Helicos Biosciences Corp. uses TRUE SINGLE MOLECULE SEQUENCING (tSMS)™ technique (Harris et al., Science 320:106-109 (2008). The tSMS™ technique uses a library of target nucleic acids prepared by the addition of a 3′ poly(A) tail to each target nucleic acid. The poly(A) tail hybridizes to poly(T) oligonucleotides anchored on a glass cover slip. The poly(T) oligonucleotide can be used as a primer for the extension of a polynucleotide complementary to the target nucleic acid.

Sequencing platforms implementing real-time monitoring of DNA polymerase activity can be used. For example, nucleotide incorporations can be detected through fluorescence resonance energy transfer (FRET) interactions between a fluorophore-bearing polymerase and γ-phosphate-labeled nucleotides as described, for example, in U.S. Pat. Nos. 7,329,492 and 7,211,414. Nucleotide incorporations can also be detected with zero-mode waveguides as described, for example, in U.S. Pat. No. 7,315,019 and using fluorescent nucleotide analogs and engineered polymerases as described, for example, in U.S. Pat. No. 7,405,281 and US 2008/0108082. The illumination can be restricted to a zeptoliter-scale volume around a surface-tethered polymerase such that incorporation of fluorescently labeled nucleotides can be observed with low background (Levene et al., Science 299:682-686 (2003); Lundquist et al., Opt. Lett. 33:1026-1028 (2008); Korlach et al., Proc. Natl. Acad. Sci. USA 105:1176-1181 (2008).

Single-molecule, real-time (SMRT™) DNA sequencing technology is described in U.S. Pat. Nos. 7,181,122, 7,302,146, and 7,313,308. SMRT chips and similar technology can be used in association with nucleotide monomers fluorescently labeled on the terminal phosphate of the nucleotide (Korlach et al., Nucleosides, Nucleotides and Nucleic Acids, 27:1072-1083, 2008). The label is cleaved from the nucleotide monomer on incorporation of the nucleotide into the polynucleotide. Accordingly, the label is not incorporated into the polynucleotide, increasing the signal:background ratio.

(d) Multiplexing

As already described, some amplification methods amplify multiple nucleic acids in parallel. Sequencing reactions can also be carried out in multiplex formats such that multiple different nucleic acid targets are manipulated simultaneously. For example, different nucleic acid targets can be treated in a common reaction vessel or on a surface of a particular substrate. This allows convenient delivery of sequencing reagents, removal of unreacted reagents and detection of incorporation events in a multiplex manner. Nucleic acid targets can also be in an array format in a spatially distinguishable manner. The target nucleic acids can be bound by direct covalent attachment, attachment to a bead or other particle or binding to a polymerase or other molecule that is attached to the surface. The array can include a single copy of a nucleic acid target at each site (also referred to as a feature) or multiple copies having the same sequence can be present at each site or feature.

In deep sequencing a plurality of related or identical nucleic acids are attached to the surface of a reaction platform (e.g., flow cell, microarray, and the like) (see e.g., Bentley et al., Nature 2008, 456:53-59). The attached DNA molecules can be amplified in situ and used as templates for synthetic sequencing (i.e., sequencing by synthesis) using a detectable label (e.g. fluorescent reversible terminator deoxyribonucleotide). Representative reversible terminator deoxyribonucleotides include 3′-O-azidomethyl-2′-deoxynucleoside triphosphates of adenine, cytosine, guanine and thymine, each labeled with a different recognizable and removable fluorophore, optionally attached via a linker. When fluorescent tags are employed, after each cycle of incorporation, the identity of the inserted base may be determined by excitation (e.g., laser-induced excitation) of the fluorophores and imaging of the resulting immobilized growing duplex nucleic acid. The fluorophore, and optionally linker, can be removed by conventional methods, thereby regenerating a 3′ hydroxyl group ready for the next cycle of nucleotide addition.

IV. Determining a Consensus Sequence

The present methods can be used to provide a consensus sequence of at least part of the adjacent segment in a nucleic acid target from a population of raw target sequencing reads of the target. If an initial population of raw target sequences do not all contain the same anchor segment, the population can be sorted to give a population of raw target sequencing reads in which part of the sequencing read is of the same anchor segment. The members of this population are then evaluated for accuracy of sequencing of the anchor segment. Members of the population in which the accuracy at least reaches (and preferably exceeds) a threshold value are carried forward for subsequent consensus sequence determination. The raw target sequencing reads carried forward are designated in a class of accepted raw sequencing reads and can be literally or conceptually assigned to an accepted class. This class is usually in the form of stored information in computer system. Members of the population failing to reach the threshold value are typically discarded and not further used in the analysis. The threshold value can be based on the percentage sequence identity between the segment of a raw target sequencing read and corresponding known sequence of an anchor segment and/or the location of matched and mismatched nucleotides between. Sequence identity is preferably determined over the full length of the known sequence of the anchor segment maximally aligned with the raw target sequencing read. Sequence identity is scored as the number of matched nucleotides divided by the number of nucleotides in the anchor segment. The sequence identity is preferably at least 80, 85, 90, 95, 99 or 100%. The threshold can additionally or alternatively be defined by the location of matched nucleotides. The nucleotide immediately adjacent to the first nucleobase of the portion of the sequencing read corresponding to the adjacent segment is particularly significant. Thus, the threshold can require this nucleobase to be accurately determined. The threshold can require a contiguous segment of at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleobases adjacent the first nucleobase of corresponding to the adjacent segment to be correctly determined.

Having selected a subset of raw target sequencing reads in which the accuracy of the portion of the read corresponding to an anchor segment exceeds a threshold, a consensus of the portions of the sequencing reads corresponding to the adjacent segment can be determined by a method including any or all of the polling, discarding or reassigning steps described below.

Preferred methods assign successive nucleobases to a nascent sequence of the adjacent sequence by a process referred to as polling. Polling compares nucleobases occupying a corresponding position among raw target sequencing reads to determine a consensus nucleobase at the corresponding position. A consensus nucleobase is the most represented nucleobase in the different raw target sequencing reads being polled at the polled position. If two or more nucleobases are tied for most represented, any of the tied bases can be regarded as the consensus nucleobase. The other tied nucleobases can be treated as non-consensus or can be treated as potential sites of sequences variations as discussed in more detail below. Nucleobases that are not tied, but less represented, can also be treated as potential sites of sequences variations. In most cases, the sites for sequence variations are not tied bases. The position for the initial polling step is defined by reference to the anchor segment so the position is equidistant from the sequence of the anchor segment in the raw target sequencing being polled. Typically the position is immediately adjacent the sequence of the anchor segment in the direction in which the sequencing read is performed (typically beginning at the adapter segment and moving into the adjacent segment). Polling determines the consensus nucleobase at this position and this nucleobase is assigned as a nucleobase of a sequence of the adjacent segment, typically the first base. Raw target sequencing reads having the consensus nucleobase at the position polled are retained as accepted sequences. Raw target sequencing reads lacking the consensus nucleobase at the position polled are designated as rejected raw target sequencing reads. The rejected sequencing reads can literally or conceptually be assigned to a rejected class. The rejected class, like the accepted class, is typically electronic information in computer memory. At this point, some rejected raw target sequencing reads may be reassigned as accepted sequences in a process that will be described in more detail below.

For raw target sequencing reads polled in the previous step and retained as accepted sequences, a further polling step is performed on the next nucleobase (adjacent the nucleobase polled in the previous step). The directionality is usually the same as that in which the raw target sequencing read is developed beginning at or before the anchor segment and moving into the adjacent segment. Thus, for a sequencing-by-synthesis method, the raw target sequencing read is determined in a 5′-3′ direction and nucleobases in successive polling steps also usually move along the raw targeting sequences in a 5′-3′ direction. However, successive polling steps can also be performed in the opposite directed to that of synthesis (i.e., 3′-5′). Again, a consensus nucleobase is determined at a corresponding position between the accepted raw target sequencing reads, and this nucleobase is assigned as the next nucleobase of the nascent sequence of the adjacent segment. Again, raw target sequencing reads having the consensus nucleobase at the polled position are retained as accepted sequences. Raw target sequencing reads lacking the consensus nucleobase at the polled position are designated as rejected sequences.

Further iterations of the poling step can be performed. A repetition polls a position adjacent the position polled in the previous polling step for accepted raw target sequencing read. The determined consensus nucleobase in successive repetitions form successive nucleobases in the nascent segment of the adjacent segment. After a polling step, raw target sequencing reads having the consensus nucleobase at the polled position are retained as accepted and raw target sequencing reads lacking the consensus nucleobase are designated as rejected.

As mentioned above, in any cycle of the above methods, rejected raw target sequencing reads can be considered for reassignment as accepted raw target sequencing reads. Usually, reassignment, if performed, occurs after the polling step and after any raw target sequencing reads not having the consensus nucleobase in the polling step are assigned as rejected sequencing reads. Reassignment allows the consensus sequence determination to make use of information from raw target sequencing reads that do not conform to the consensus sequence of the adjacent segment in one region but do in one or more other regions. Such lack of conformity may be the result of a sequencing error, a polymorphism, deletions of part of the adjacent segment of the nucleic acid target or a heterogeneous mixture of nucleic acid targets in which adjacent segments do not necessarily begin and end at the same point of a large molecule. Rejected raw target sequencing reads are reassigned based on overall sequence identity with the nascent sequence and/or the location of matched nucleobases between the rejected raw target sequencing read and the nascent sequence. A rejected raw target sequencing read is aligned with the nascent sequence to maximize matched nucleotides. The alignment can be performed over the entire length of the nascent sequence or a window of e.g., the last 10 or 20 nucleobases. Percentage sequence identity can be calculated as the number of matched nucleobases with the nascent sequence minus deletions or insertions divided by the number of nucleobases of the nascent sequence. The calculation can be performed over the entire nascent sequence or a window thereof, for example, the last 20 or 10 nucleobases. The threshold for sequence identity can be defined as at least 80, 85, 90, 95, 99 or 100% over the defined window. Additionally, or alternatively, the threshold can require identity between the last nucleotide determined for the nascent sequence and the corresponding nucleotide of the rejected raw target sequence. The threshold can alternatively require identity between the last 2, 3, 4, 5, 6, 7, 8, 9 or 10 determined nucleotides of the nascent sequence and the corresponding nucleotides of the raw target sequencing read. An exemplary criterion for reassigning a rejected raw target sequencing read to be an accepted raw target sequencing read is an overall sequence identity of at least 80% and identity between the last determined nucleobase of the nascent sequence and corresponding nucleobase of the raw target sequencing read.

Rejected raw targeting sequencing reads can be assessed for reassignment to accepted status in any iteration of the method. The fact that rejected raw targeting sequencing are assessed may or may not result in one or more of them being found to meet the threshold for reassignment to accepted status. In some methods, a reassignment is made in at least one cycle. In some methods, a reassignment is performed at least 5 times in at least 20 cycles. In some methods, a reassignment is performed at least 20 times in at least 100 cycles. Reassignment or at least assessment of rejected raw target sequencing reads for reassignment can also performed at regular intervals, e.g., every 5 or 10 polling steps, or after the first 20 polling steps, and then after each 5 or ten polling steps thereafter.

The interval between a raw target sequencing read being assigned from the accepted to rejected class and then back to accepted can be as short of one cycle. For example, a raw target sequencing read having a single nucleobase insertion can be assigned from accepted to rejected because the inserted nucleobase is not the consensus nucleobase at the relevant position being polled. However, the same raw target sequencing read can then be immediately reassigned because its next nucleobase is the last polled nucleobase in the consensus sequence and it overall meets the threshold criteria for reassignment to accepted status. In this case, the raw target sequence read effectively misses only one nucleobase being read corresponding to the nucleobase insertion. For raw target sequencing reads having a single nucleobase substitution, when the nucleobase occupied by the substitution is polled, the raw target sequencing read does not have the consensus nucleobase and is assigned to the rejected class. However, after the next round of polling, the raw target sequencing read does have a nucleobase the same as the most recently determined nucleobase of the nascent sequence and can be returned to the accepted class (assuming other threshold criteria are met). In this case, the raw target sequencing read has effectively missed two nucleobases being read in determining the consensus sequence, the substituted nucleobase and the next adjacent nucleobase. Other raw target sequencing reads are present in the rejected class of sequences for longer periods or may never be returned to the accepted raw target sequencing reads.

Returning raw target sequencing reads either were not polled in the previous polling cycle, of if polled, yielded a non-consensus nucleobase. In the polling cycle immediately after a raw targeting sequencing read is returned to the accepted class of sequencing reads, the position polled is that immediately adjacent to the position aligned with the last nucleobase determined in the nascent sequence so as to permit assessment of the next nucleobase in the nascent sequence Immediately returning raw sequencing reads are polled together with raw target sequences already having accepted status to determine a consensus nucleobase. If a returning raw target sequencing read remains in the accepted category following its initial return, positions for subsequent polling can be determined as for other raw targeting reads in the accepted category. That is, the position for one polling cycle is the position adjacent that in the previous polling cycle preserving a directionality throughout polling such that successive nucleobases in the nascent sequence are determined. For raw sequencing reads generated in a 5′-3′ orientation, the directionality of polling successive nucleobases is also usually 5′-3′ but can also be 3′-5′.

The steps of polling, and assigning raw target sequencing reads are continued assigning successive nucleobases to the nascent sequence until a sufficient length of nascent sequence has been determined or the complete length of the adjacent segment has been determined or the number of raw target sequencing reads in the accepted class falls below a threshold limit. The accuracy of raw target sequencing reads typically reduces further along the read. As the accuracy is reduced, more raw target sequencing reads are designated as rejected sequencing reads and fewer, if any, are returned to the accepted class. The number of cycles of polling (and auxiliary assigning and reassigning steps) depends on the length of the adjacent segment and the length of reasonably accurate sequencing read, which in turn depends on the sequencing technique. Depending on the sequencing technique, the number of polling cycles can be e.g., at least 2, 5, 10, 50, 100, 200, 1000, 10,000 or 50,000.

The process described above identifies the consensus nucleobases occupying successive positions of the nascent sequence. The process can be varied or extended to determine variants of the consensus sequence as well. The variations can be allelic variations, variations between isolates, strains or species, or sequence variations between a population of viral molecules in a clinical sample, among others. Such variations can be identified by forming branched consensus sequences as the method described above is performed or by repeating the method on raw target sequencing reads that have been rejected at one or more cycles of the method. Branching starts by identifying two (or more) consensus nucleobases in a polling step. The nucleobases may have the same or similar representation in the accepted raw target sequencing reads, or one nucleobase may have higher representation than the other but both nucleobases still have a representation exceeding a threshold. In this situation, the nascent sequence is branched into two nascent sequences with the two consensus nucleobases being the first nucleobases in the two branched arms of the nascent sequence. Raw target sequencing reads having either of the consensus nucleobases are retained in the accepted class. Subsequent consensus nucleobases are assigned to both branched nascent sequences. A branched nascent sequence can itself be subject of further branching at a further position of sequence variation.

Alternatively of additionally, a consensus sequence of the adjacent segment can be determined without branching and the process repeated using raw targeting sequences that have been rejected in at least one cycle and preferably returned to the accepted class subsequently. These sequences are a likely source of sequence variants because a raw targeting sequencing read will be rejected if it includes one or more nucleobase differing from the consensus but can then be returned to the accepted class based on identity between one or more subsequently determined nucleobases and the consensus nucleobase. Performing further iterations of the method on raw targeting sequencing reads that have returned to the accepted class can thus be used to identify one or more variants of the initially determined consensus sequence of the adjacent segment.

Once the sequence of an unknown adjacent segment has been determined by repeated polling and discarding subreads as described above, the process can be repeated starting with raw target sequencing reads in the rejected class after performing the process. If the initial population of raw target sequencing reads including sequencing reads of multiple nucleic acid targets includes the same anchor segment linked to different adjacent segments, repeating the method can be used to determine the sequence of a different adjacent segment. The method initially determines the sequence of the predominant adjacent segment in such a mixture, with nucleic acid targets containing other adjacent segments being designated as rejected sequences. Repeating the method on the rejected sequences, thus allows determination of a consensus sequence for a second and different adjacent segment. The method can be repeated multiple times to determine the consensus sequence of multiple different adjacent segments.

An initial population of raw target sequencing reads sometimes includes sequencing reads of nucleic acid target incorporating different anchor segments. Different anchor segments can be used to distinguish reads of opposing strands of an adjacent segment or different adjacent segments. If different segments, the segments can be overlapping or part of the same larger nucleic acid molecule (e.g., a genome or chromosome). In this case, the raw target sequencing reads can usually be segregated by anchor segment so as to be in groups in which the read is that of the same anchor segment. The above methods can be applied separately to the raw target sequencing reads in the same group.

In a further variation, after determining a consensus sequence of an adjacent segment, part of the consensus sequence is itself used as an anchor segment in an additional iteration of the method. The additional iteration can start with sequences in the rejected class and/or any sequences remaining in the accepted class. The adjacent segment for the new anchor segment can overlap in full, in part or not at all with adjacent segment from the first iteration. If the adjacent segment overlaps completely, then the additional iteration provides a check and possible identification and correction of errors in the consensus sequence. If the adjacent segment is completely beyond the prior adjacent segment, then an additional consensus sequence is determined contiguous with the consensus sequence initially determined. If the additional adjacent segment is partially within and partially beyond the prior adjacent segment, then it both checks and extends the consensus read from the prior adjacent segment.

The result of the above method is at least a consensus sequence of a part of an adjacent segment. Sometimes alternative consensus sequences including a sequence variation are also provided. Sometimes consensus sequences for both strands of an adjacent segment are provided. Sometimes consensus sequences of multiple adjacent segments are provided. If consensus sequences are provided for both strands of an adjacent segment and there are any discrepancies, the discrepancies can be rechecked, optionally using an alternative sequencing method. As already noted forward and reverse sequences can be readily generated using certain sequencing platforms such as SMRT technology. Discrepancies sometimes arise from reading a particular nucleobase but not its complementary nucleobase. If consensus sequences are provided for multiple adjacent segments that are part of the same larger nucleic acid molecule the sequences can be combined based on overlaps by conventional methods.

Determining sequence variations requires distinguishing between sequencing errors and true sequence variations. Such a distinction can be made by setting certain filtering criteria, or by setting a rank threshold such as a quality score. One example of a filter for identifying error or variant at any given position is to quantify the number of times each nucleobase appeared at a given position. The nucleobase that occurs the majority of the time is likely the correct residue for that position. For the remaining non-majority nucleobases that appeared at a given position, if their occurrence is relatively even meaning about 33% for each, then is can be determined that for this given position, the mismatches were errors. On the other hand, should one of the remaining non-majority nucleobases appear more frequently than the others, then that nucleobase is likely the correct nucleobase of a minority species variant. Of course, quantifying the relative occurrence of the non-majority nucleobases should take into account the statistical significance of any differences in occurrences. An example of a quality metric is a Phred score (Hillier et al., Genome Res. 8(3):175-185, 1998). for Sanger sequencing, which is calculated based on fluorescent signal characteristics for the 4 nucleobase channels at a given position. A high Phred score for a predominant non-majority nucleobase at a given position would be an indication that the variant is present in a legitimate minority species. It is often useful to calculate a log odds ratio based on quality scores for each potential nucleobase. The log odds ratio is the natural log of the ratio of odds that a nucleobase is present based on experimental data, and represents the likelihood that a particular nucleobase was correctly read at a given position. Thus, a high log odds ratio for the predominant non-majority nucleobase at a given position suggests that it is a valid nucleobase at that position in a legitimate minority species.

Sequencing errors and true sequence variations can be further distinguished by comparing determined sequences of multiple adjacent segments. For example, adjacent segments covering different but partially overlapping regions of a larger nucleic acid molecule suspected of having the sequence variations can be compared. True sequence variations are more likely to appear in most, if not all, partially overlapping sequences. Special care should be taken, however, when phasing multiple SNPs within chromosomes. For example, when phasing multiple SNPS within chromosomes, it is possible that all SNPs are located in only one allele. In these cases, data generated from each allele should not be combined. Preferably, determined sequences from each allele should be compared against each other to phase multiple SNPs.

FIG. 1 provides an overview of an exemplary analysis schema for analyzing a target nucleic acid. At step 1, raw target sequencing reads were selected to create a class of raw target sequencing reads having high-quality reads of the anchor segment. Different criteria can be applied in extracting such raw target sequencing reads. For examples, raw target sequencing reads preferably include only reads having sequences that are 100% identical to the known, correct anchor segment sequence. Optionally, reads can be filtered to create a class of raw target sequencing reads having sequences that are at least 99%, 95%, 90%, 85%, 80%, 75%, or 70% identical to the known, correct anchor segment sequence. Filtering criteria are used for evaluating the accuracy of the sequencing, and can be adjusted based on various parameters, e.g., the number of subreads having high-quality reads of the anchor segment. Raw target sequencing reads that meet pre-determined criteria are accepted for base-polling (see step 2 of FIG. 1).

VII. Computer Implementation

The present methods can be computer-implemented, such that at least one or more (e.g., at least 2, 3, 4, 5, 6, 7, or 8), or all steps of the method are carried out by a computer program (except wet chemical steps). The present methods can be implemented in a computer program stored on computer-readable media, such as the hard drive of a standard computer. A computer program for analyzing a nucleic acid target can include one or more of the following codes: (a) code for receiving a population of raw target sequencing reads of a nucleic acid target comprising an anchor segment and an adjacent segment, (b) code for evaluating the accuracy of sequencing of the anchor segment in different raw target sequencing reads by comparing raw target sequencing reads of the anchor segment with the known sequence of the anchor segment, (c) code for assigning a subset of the raw target sequencing reads into an accepted class based on reaching at least a threshold level of accuracy of the sequencing of the anchor segment, (d) code for polling nucleobases at a position equidistant to the anchor segment sequence in raw target sequencing reads in the accepted class to determine a consensus nucleobase, which consensus nucleobase is assigned as the first nucleobase of a nascent sequence of the adjacent segment, (e) code for assigning raw target sequencing reads having the consensus nucleobase determined in the prior polling step to remain in an accepted class and assigning raw target sequencing reads lacking the consensus nucleobase determined in the prior polling step to the rejected class, (f) code for optionally reassigning a raw target sequencing read from the rejected class to the accepted class by scoring similarity of the raw target sequencing read to the nascent sequence and reintroducing the raw target sequencing read if the sequence similarity reaches at least a threshold level of similarity, and code for repeating steps coded in (d), (e) and optionally (f), except that a repetition polls a position adjacent the position poled in the previous polling step for raw target sequencing reads having the consensus nucleobase polled in the previous step or in the case of a raw target sequencing read reassigned from the rejected class to the accepted class and not polled in the previous polling step or if polled not having the consensus nucleobase in the previous polling step, the polling polls a position adjacent the position aligned with the last nucleobase of the nascent sequence to determine a consensus nucleobase, and the consensus nucleobases determined in successive repetitions are assigned as successive nucleobases in the nascent sequence of the adjacent segment. A computer program for analyzing a nucleic acid target can also include one or more of the following codes: code for receiving a population of raw target sequences of a nucleic acid target comprising an adapter segment and an adjacent segment, code for evaluating the accuracy of sequencing of the adapter segment in different raw target sequences by raw target sequencing reads of the anchor segment with the known correct sequence of the anchor segment, code for assigning a subset of the raw target sequences into an accepted class based on the accuracy of sequencing of the adapter segment in the raw target sequences, code for aligning at least some of the raw target sequences from the accepted class, code for determining a sequence of at least part of the adjacent segment from the aligned sequences, and a computer-readable storage medium comprising the codes.

The present methods can be implemented in a system (e.g., a data processing system) for analyzing a nucleic acid target. The system can also include a processor, a system bus, a memory coupled to the system bus, wherein the processor is coupled to the system bus for one or more of the following: (a) receiving a population of raw target sequencing reads of a nucleic acid target comprising an anchor segment and an adjacent segment, (b) evaluating the accuracy of sequencing of the anchor segment in different raw target sequencing reads by comparing raw target sequencing reads of the anchor segment with the known sequence of the anchor segment, (c) assigning a subset of the raw target sequencing reads into an accepted class based on reaching at least a threshold level of accuracy of the sequencing of the anchor segment, (d) polling nucleobases at a position equidistant to the anchor segment sequence in raw target sequencing reads in the accepted class to determine a consensus nucleobase, which consensus nucleobase is assigned as the first nucleobase of a nascent sequence of the adjacent segment, (e) assigning raw target sequencing reads having the consensus nucleobase determined in the prior polling step to remain in an accepted class and assigning raw target sequencing reads lacking the consensus nucleobase determined in the prior polling step to the rejected class, (f) optionally reassigning a raw target sequencing read from the rejected class to the accepted class by scoring similarity of the raw target sequencing read to the nascent sequence and reintroducing the raw target sequencing read if the sequence similarity reaches at least a threshold level of similarity, and repeating steps (d), (e) and optionally (f), except that a repetition polls a position adjacent the position poled in the previous polling step for raw target sequencing reads having the consensus nucleobase polled in the previous step or in the case of a raw target sequencing read reassigned from the rejected class to the accepted class and not polled in the previous polling step or if polled not having the consensus nucleobase in the previous polling step, the polling polls a position adjacent the position aligned with the last nucleobase of the nascent sequence to determine a consensus nucleobase, and the consensus nucleobases determined in successive repetitions are assigned as successive nucleobases in the nascent sequence of the adjacent segment. The system can also include a processor, a system bus, a memory coupled to the system bus, wherein the processor is coupled to the system bus for one or more of the following: receiving a population of raw target sequences of a nucleic acid target comprising an adapter segment and an adjacent segment, evaluating the accuracy of sequencing of the adapter segment in different raw target sequences by comparing raw target sequencing reads of the anchor segment with the known correct sequence for the anchor segment, assigning a subset of the raw target sequences into an accepted class based on the accuracy of sequencing of the adapter segment in the raw target sequences, aligning at least some of the raw target sequences from the accepted class, and determining a sequence of at least part of the adjacent segment from the aligned sequences.

Various steps of the present methods can utilize information and/or programs and generate results that are stored on computer-readable media (e.g., hard drive, auxiliary memory, external memory, server; database, portable memory device (e.g., CD-R, DVD, ZIP disk, flash memory cards), and the like. For example, information used for and results generated by the methods that can be stored on computer-readable media include raw target sequencing reads of a nucleic acid target, the sequence of an anchor segment, the accepted class, the rejected class, the nascent sequence of the adjacent segment, the threshold level of similarity, the threshold level of accuracy of the sequencing the anchor segment, and one or more consensus nucleobase(s). Information used for and results generated by the methods that can be stored on computer-readable media also include raw target sequences of a nucleic acid target, the adapter segments, the accepted class, the partially or fully determined sequences of the unknown segments (i.e., the nucleobases in the adjacent segment adjacent the adapter segment), the discarded raw target sequences, the discarded raw target sequences reassigned to the accepted class, the sequence variations at each position.

The present invention also includes an article of manufacture for analyzing a nucleic acid target that includes a machine-readable medium containing one or more programs which when executed implement the steps of the present methods.

FIG. 2 is a block diagram showing a representative example of a configuration of a device for analyzing a nucleic acid target in which various aspects of the invention may be embodied. The invention can be implemented in hardware and/or software. For example, different aspects of the invention can be implemented in either client-side logic or server-side logic. The invention or components thereof can be embodied in a fixed media program component containing logic instructions and/or data that when loaded into an appropriately configured computing device cause that device to perform according to the invention. A fixed media containing logic instructions can be delivered to a viewer on a fixed media for physically loading into a viewer's computer or a fixed media containing logic instructions may reside on a remote server that a viewer accesses through a communication medium in order to download a program component.

FIG. 2 shows an information appliance (or digital device) that may be understood as a logical apparatus that can read information (e.g., instructions and/or data) from auxiliary memory 212, which may reside within the device or may be connected to the device via, e.g., a network port or external drive. Auxiliary memory 212 can reside on any type of memory storage device (e.g., a server or media such as a CD or floppy drive), and can optionally comprise multiple auxiliary memory devices, e.g., for separate storage of raw target sequences, determined sequences of the segments adjacent the adapter sequence, sequence variations information, and/or other information. The device can thereafter use that information to direct server or client logic to embody aspects of the invention.

One exemplary type of logical apparatus is a computer system as illustrated in FIG. 2, containing a CPU 201 for performing calculations, a display 202 for displaying an interface, a keyboard 203, and a pointing device 204, and further comprises a main memory 205 storing various programs and a storage device 212 that can store the raw target sequencing reads of a nucleic acid target 213 and the nascent sequence of the adjacent segment 214 and the consensus nucleobase 215. The device is not limited to a personal computer, but can be any information appliance for interacting with a remote data application, and can include such devices as, for example, a digitally enabled television, cell phone, or personal digital assistant. Information residing in the main memory 205 and the auxiliary memory 212 can be used to program such a system and can represent a disk-type optical or magnetic media, magnetic tape, solid state dynamic or static memory, or the like. For example, the invention may be embodied in whole or in part as software recorded on this fixed media. The various programs stored on the main memory can include a program to receive a population of raw target sequences of a nucleic acid target, a program 206 to receive a population of raw target sequencing reads of a nucleic acid target comprising an anchor segment and an adjacent segment, a program 207 to evaluate the accuracy of sequencing of the anchor segment in different raw target sequencing reads by comparing the anchor segment of a raw target sequencing read with the known sequence for the anchor segment, a program 208 to assign a subset of the raw target sequencing reads into an accepted class based on reaching at least a threshold level of accuracy of the sequencing of the anchor segment, a program 209 to poll nucleobases at a position equidistant to the anchor segment sequence in raw target sequencing reads in the accepted class to determine a consensus nucleobase, a program 210 to assign raw target sequencing reads having the consensus nucleobase determined in the prior polling step to remain in an accepted class and assigning raw target sequencing reads lacking the consensus nucleobase determined in the prior polling step to the rejected class, and a program 211 to reassign a raw target sequencing read from the rejected class to the accepted class by scoring similarity of the raw target sequencing read to the nascent sequence and reintroducing the raw target sequencing read if the sequence similarity reaches at least a threshold level of similarity. The lines connecting CPU 201, main memory 205, and auxiliary memory 212 can be any type of communication connection.

Raw target sequences and parameters required for the present methods can be specified by the display 202 (also referred to as a “screen”), the keyboard 203, and the pointing device 204. The CPU 201 can then execute a program stored in the main memory 205 and the sequence of a segment adjacent the adapter sequence including sequence variations, if present, can be determined by the present methods. The raw target sequencing reads of a nucleic acid target 213 can be read from the storage device 212. The output result of the nascent sequence of the adjacent segment 214 and the consensus nucleobase 215 can be stored into the storage devices 212. The progress of this processing can be displayed on the display 202. After completing this processing, the result of the processing can be also displayed on the display 202, saved to an additional storage device (e.g., ZIP disk, CD-R, DVD, floppy disk, flash memory card), or displayed and/or saved in hard copy (e.g., on paper). The result of the processing can be stored or displayed in whole or in part, as determined by the user.

VIII. Applications

The nucleic acid target or adjacent segment thereof can be derived from any of a number of sources, for example, viruses, prokaryotes, or eukaryotes (e.g., plants, fungi, and animals). These sources can include biological samples including patient and environmental samples (agricultural, water, soil), research samples, and industrial samples. A biological sample is a composition or mixture in which a nucleic acid molecule of interest may be present, including plant or animal materials, waste materials, materials for forensic analysis, environmental samples, and the like. A biological sample includes any tissue, cell, or extract derived from a living or dead organism which may contain a target nucleic acid, e.g., peripheral blood, bone marrow, plasma, serum, biopsy tissue including lymph nodes, respiratory tissue or exudates, gastrointestinal tissue, urine, feces, semen, or other body fluids. Samples of particular interest are patient tissue samples (including body fluids) from a human or an animal having or suspected of having a disease or condition, particularly infection by a virus. The nucleic acid target of interest in a patient sample can be from a pathogenic microorganism, such as a virus, bacteria or fungus, or can be endogenous to a patient, or both types of target can be of interest. Other samples of interest include industrial samples, such as for water testing, food testing, contamination control, and the like. Sample components may include nucleic acids to be sequenced and other nucleic acids, and other materials such as salts, acids, bases, detergents, proteins, carbohydrates, lipids and other organic or inorganic materials.

Nucleic acid targets or adjacent segments thereof can be isolated from samples using any of a variety of conventional procedures, for example target capture using a target-capture oligomer and a solid support (e.g., U.S. Pat. No. 6,110,678, EP 1778867, WO 2008/016988 & WO 2009/140374), the Applied Biosystems ABI Prism™ 6100 Nucleic Acid PrepStation, and the ABI Prism™ 6700 Automated Nucleic Acid Workstation, Boom et al., U.S. Pat. No. 5,234,809, or mirVana RNA isolation kit (Ambion). Nucleic acids can be cut or sheared prior to analysis, including the use of such procedures as mechanical force, sonication, restriction endonuclease cleavage, or other conventional methods to produce nucleic acid targets or adjacent segments from which nucleic acid targets can be formed.

The nucleic acid target or adjacent segment thereof can be DNA (genomic or cDNA), RNA (e.g., viral RNA, micro RNA, mRNA, cRNA, rRNA, hnRNA, transfer RNA, siRNA), and can comprise nucleic acid analogs or other nucleic acid mimics subjectable to sequence determination. The nucleic acid target or adjacent segment thereof can also be fragmented genomic DNA (gDNA), micro RNAs (miRNAs) or other short RNAs, or a short target nucleic acid is a short DNA molecule derived from a degraded source, such as can be found in for example forensics samples (see for example Butler, 2001, Forensic DNA Typing: Biology and Technology Behind STR Markers). The target can be methylated, non-methylated, or both. The target can be bisulfite-treated and have non-methylated cytosines converted to uracil.

A target nucleic acid can be synthetic or naturally occurring. Reference to a nucleic acid target can mean the nucleic acid target itself or s surrogates thereof, for example amplification products.

The present methods can be used in various applications, for example, de novo sequencing, DNA fingerprinting, polymorphism identification (e.g., SNPs) or other nucleic acid analysis. One application is determining the sequences of a heterogeneous population of variant nucleic acid molecules such as variant nucleic acid molecules of a same virus (e.g., HIV or HCV). Some examples of viruses that can be detected include HIV, hepatitis (A, B, or C), herpes virus (e.g., VZV, HSV-1, HAV-6, HSV-II, CMV, and Epstein Barr virus), adenovirus, XMRV, influenza virus, flaviviruses, echovirus, rhinovirus, coxsackie virus, cornovirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, MLV-related Virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus and arboviral encephalitis virus.

Analysis of viral nucleic acids is particularly useful for analyzing drug resistance and the emergence of drug resistant viral strains presenting as minor variants in a virus population. Viruses mutate rapidly so that a patient is often infected with a heterogeneous population of viral nucleic acids, which changes over time. Some of the mutations differentiating species of the heterogeneous population may be associated with resistance to a drug that the patient has been treated with or may be treated with in the future. Deconvolution of the population to detect individual variants allows detection of drug resistant mutations and their change over time, thus allowing treatment regimes to be customized to take into account the drug resistance of strains infecting a particular patient. Because drug-resistant or other mutations may present as only a small proportion of viral nucleic acid molecules, sequencing of a large number of molecules in the viral nucleic population may be required to provide a high likelihood of identifying all drug resistant mutations or at least all, whose representation as a percentage of the total viral nucleic acid population exceeds a threshold.

The present methods can also be used for detecting SNP and somatic mutations. For example, the methods can be used to detect and characterize rare variants and identify unknown causative mutations in human diseases. The improved detection of rare sequence variants by the methods of the invention can also be applied to the discovery of novel somatic mutations, e.g., in cancers. Comprehensive genomic analysis of a variety of cancers can be performed, including acute myeloid leukemia, lung cancer, and melanoma. The present methods can be used to detect expression products of specific alleles, haplotype analysis and phasing of multiple SNPs within chromosomes, and copy number variation of DNA segments.

Human nucleic acids are useful for diagnosing diseases or susceptibility towards disease (e.g., cancer gene fusions, BRACA-1 or BRAC-2, p53, CFTR, cytochromes P450), for genotyping (e.g., forensic identification, paternity testing, heterozygous carrier of a gene that acts when homozygous, HLA typing), determining drug efficacy on an individual (e.g., companion diagnostics) and other uses. Sequence variations information obtained from the present methods can be used to treat the subjects differentially. For example, samples from members of the patient population can be sequenced. The sequencing can provide information about a pathogenic microorganism infecting a patient (for example, type of organism and/or drug resistance). The sequencing can alternatively or additionally provide information about a patient gene associated with genetic disease, susceptibility or response to infection or response to treatment. Different members of the patient population can receive different treatment regimes (including no treatment) depending on the determined sequence for the sample from each member.

The present methods can also be used for epigenetics studies. For example, the methods can be used for detecting DNA methylation, such as aberrant methylation associate with various diseases such as cancers. The methods can also be used to select patients for demethylation therapies and to monitor the therapeutic response to demethylation agents.

The present methods can also be used for RNA analysis. Analysis of rRNA is particularly useful for detecting and/or typing pathogenic bacteria. Examples of such bacteria include chlamydia, rickettsial bacteria, mycobacteria, staphylococci, treptocci, pneumonococci, meningococci and conococci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague, leptospirosis, Lymes disease bacteria, streptococci, or neisseria. Ribosomal RNAs in these various organisms typically have conserved sequences and variant sequences that are unique to one or a few different organisms. A conserved sequence can be used to identify an rRNA and a variant sequence to identity an organism of which the variant sequence is characteristic. For example, U.S. Pat. No. 7,226,739 and U.S. Pat. No. 5,541,308 disclose conserved and variable rRNA sequences in a plurality of bacteria. Similarly, many diseases are associated with aberrant mRNA expression. The present methods can be used for transcriptome analysis (RNA-seq) such as small RNA mapping and transcriptome mapping.

Nucleic acids having sequences determined by present methods can be synthesized by conventional methods, including solid state synthesis and primer extension.

EXAMPLES Example 1 Sequence Determination by Base-Polling

FIGS. 4A-D provides an illustrative example of sequence determination using the base-polling methods as described in the present invention. The example illustrates the sequence determination algorithm using an initial set of 9 raw target sequences (SEQ ID NOs:1-9). Raw target sequences that meet certain criteria (e.g., sequences being polled or reassigned) were placed into an “Accepted” class, and those that fail the same criteria were placed into a “Rejected” class. For illustration purposes, a total of four iterations of base-polling are provided for determining a sequence of four nucleotides.

Iteration 1

Raw target sequences 1-9 were chosen based on the quality of an adapter sequence (not shown), and aligned over the region of the adapter sequence. These sequences were placed into the accepted class and were used as the initial population of raw target sequences for base-polling. As illustrated in Iteration 1, the dominant nucleobase at the first nucleobase position is nucleotide C. The raw target sequences were accordingly polled and the first nucleobase of the sequence determined is C. Sequences 1-4, and 6-9, having C as the first base, remain in the accepted class. Sequence 5, having a T at the first base, were placed in the rejected class. The vertical bar in the accepted set indicates that the sequence segment before the bar is the sequence determined so far. The nucleobase after the vertical bar would be the next nucleobase for polling.

The sequences in the rejected class (e.g., sequence 5) were then compared with the determined sequence (e.g., a sequence comprising the first nucleobase C). In Iteration 1, sequence 5 was not reassigned into the accepted class because the first nucleobase of in sequence 5 is not the first nucleobase determined in the polling step, and there is not enough sequence similarity between the first nucleobase determined in the polling step and the first nucleobase of in sequence 5.

Iteration 2

The polling action at the second nucleobase generated the second polled base, T. Sequences 1, 2, and 4 were placed into the rejected class because the second nucleobase in these sequences is not T. Sequence 5 in the rejected class is carried over from the last iteration.

The sequences in the rejected class were then compared with the determined sequence CT. Both the first (C) and the second nucleobase (T) were found in sequences 4 and 5, even though they appear at the second and third positions by sequential numbering due to a single-base insertion. Therefore, sequence comparison found sequences 4 and 5 as good matches with the determined sequence CT. These two sequences were reassigned to the accepted set, leaving only sequences 1 and 2 in the rejected set.

Iteration 3

The polling action at the third nucleobase generated the third polled base, G. Sequence 7 was placed into the rejected class because the third nucleobase in sequence 7 is not G. All three sequences (1, 2, and 7) were not found to be similar to the determined sequence CTG. Sequences 1, 2, and 7 were not reassigned into the accepted class because there is not enough sequence similarity between the determined sequence CTG and these sequences.

Iteration 4

The polling action generated the fourth polled base, C. Sequences 6 and 8 were placed into the rejected class because the fourth nucleobase in these sequences is not C.

The sequences in the rejected class were then compared with the determined sequence CTGC. The fourth nucleobase C was found in these sequences, even though it appears at the third position of sequence 1 by sequential numbering due to a single-base deletion, and at the fifth positions of sequences 6 and 8 by sequential numbering due to a single-base insertion. These three sequences were reassigned to the accepted set because their overall sequences were highly similar to the consensus sequence.

Example 2 The Sample Sequence, Sequencing and the Primary Analysis Data

The sample sequenced was a region in the HCV 5′ UTR of 164 base pair long that is listed below:

(SEQ ID NO: 10) CTGCGGAACCGGTGAGTACACCGGAATTGCCAGGACGACCGGGTCCTTTC GTGGATAAACCCGCTCAATGCCTGGAGATTTGGGCGTGCCCCCGCAAGAC TGCTAGCCGAGTAGTGTTGGGTCGCGAAAGGCCTTGTGGTACTGCCTGAT AGGGTGCTTGCGAG

The PacBio's standard sample preparation and SMRT™Bell preparation methods were used.

The sequencing was carried out on a PacBio RS sequencer using the following protocols.

TABLE 1 Protocols used in sequencing and primary analyses Protocol RS_CircCons_HCv1a3bUTR.1 Collection protocol Standard Seq 2-Set v1 Primary protocol BasecallerV1

There are two videos of 45 minutes long. Data from the two videos are combined for the subsequent analyses.

TABLE 2 Per video sequencing statistics Video 1 Video 2 Reads of productivity = 0 2031 (2.7%)  5033 (6.7%)  Reads of productivity = 1 44625 (59.38%) 44098 (58.69%) Reads of productivity >1 28497 (37.92%) 26008 (34.61%) Mean Quality Score 0.79 0.8 (productivity = 1) Mean Read Length (productivity = 1398.67 1569.75 1) Pass Filter 59.73% 58.85% Active ZMWs 97.30% 93.30% IPD 0.22 0.26 Poly. Speed 2.21 2.05

TABLE 3 The combined sequencing statisticsare listed below. Total Bases 391035620 Total Reads 150292 Total Reads of productivity = 0 7064 Total Reads of productivity = 1 88723 Total Reads of productivity >1 54505 Total Active ZMWs 143228 Mean Quality Score 0.79 (productivity = 1) Mean Read Length 1483.7 (productivity = 1)

From the large number of files generated in PacBio's primary analysis, we only used the raw FastA file for all the ZMWs as our input.

On the PacBio platform, the sequenced molecule is in a SMRT™Bell format with a double stranded insert and a hairpin adapter at each ends. That produces a read of alternating forwarding and reversed strand of the insert interspersed with the adapter sequence.

The adapter sequence is

(SEQ ID NO: 11) ATCTCTCTCAACAACAACAACGGAGGAGGAGGAAAAGAGAGAGAT

Example 3 The Workflow of the Polling Algorithm

(1) Subread Extraction:

(a) Identify the adapter sequences and generate the subreads from each read.

(b) This process also offers some local sequencing quality information that can be used to further filter out low quality regions. That includes the spatial quality (the particular ZMW) and the temporal quality (the adjacent bases have a higher probability to be more similar than distant ones).

(c) Filter the subread set using certain criteria.

(2) Run Polling Algorithm:

(a) Assign all subreads with good adapter quality and sufficient length to the initial accepted set.

(b) A single base polling step: (i) Poll the most dominant base from the next base (the base immediately after the consensus matched segment of the subread in the accepted set, and initially it is the first base of the subread) of all the subreads in the accepted set. (ii) Assign the dominant base to be the next base in the growing consensus; (iii) Move the subreads with different bases to the rejected set (the newly rejected); (iv) Use the Overlap Matching pairwise-alignment algorithm that does not penalize overhanging ends to score the subreads in the rejected set with the consensus sequence. (v) Return the good matches to the accepted set (the returned).

(c) Repeat step b until a stop condition is met. The stop condition can be a pre-defined consensus length, the minimum size of the accepted set at that step, or a significant increase of the terminated subread at that step.

(3) After finishing one consensus, step 2 can be repeated with all the subreads in the rejected set. Iterate through to generate more consensuses until there is not enough subreads left.

Example 4 The Identification of the Adapters, Generation of Subreads and Quality Scores

From the primary data, the FastA files, all the adapter sequences in the raw sequence from each ZMW were identified using the algorithm described herein. We used the Overlap Matching alignment to align adapter sequence with the raw read. A score was computed from the alignment as

${Score} = \frac{{Sum}\mspace{14mu} {of}\mspace{14mu} {mismatches}\mspace{14mu} {and}\mspace{14mu} {indels}}{{The}\mspace{14mu} {length}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {matched}\mspace{14mu} {portion}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {adapter}\mspace{14mu} {sequence}}$

The score served as the quality score of the adapter. The alignment generated two unmatched fragments, occasionally one fragment from the read. The process was repeated recursively over the newly generated fragment until no more adapter matches could be found for the cutoff value 0.2 per base. In other words, for an alignment with a full-length adapter of 45 bases, the maximum allowed differences, mismatches and indels, was nine. The lower bound for the length of the matched portion of the adapter sequence was also set to be 24. Furthermore, the acceptable subreads were limited to the length between 60%-140% of the length of the amplicon that was 98-230, and there must be at least three subreads in a read.

124343 (83%) raw reads were removed due to short inserts (possibly adapter dimers) and poor quality adapters from further analyses. In the remaining 24836 reads, 179001 subreads were generated according to the adapter locations.

The matched sequences are normally not perfectly matched to the adapter sequence. For example, there can be some long regions without a good match indicating the quality was too poor for the adapter to be matched. Below is an example of removed low quality raw read. Notice the short length and long stretches like “AAAAAAAAAAAAAAAA (SEQ ID NO:12)”.

>m110510_124258_sherri_c100084032555500001215005706031134_s1_p0/54 (SEQ ID NO: 13) TGCAGCAGGGCGGCTGCTGAGAGTGATGGTCGCGACACTTGACTCGCAGGGTGACAAGAAAG CGCCTCTCCCCCATTGCCTCTTGTAAAATCCACGAGAACAAGACCGCCATCCGACCCAAACA AAAACGACACTCAAAAAACAGCCACCAAAAAAACAAGCACAGAAGCAACCAAAAGAAACCAC CAACCACACCCAGGAAAAAAAAAACAAAAAAAAACAAAAAAAAAACAAAAAAAAAACCACAC CCACACATCATCTACAAACAACAAAAAAGACCGAAAAAAAAAAAAGATCGGACCCACCACCA ATAACCTATACAACCACTTAAGAACGCGCAGCCACCCCCATCCACGAACAAAAAACACAACA GCCAAAGAACACCAAAAAAAAAAAACAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA TGGACGTGCTTGCCGAATGCGCGGTGGCGCTT

Five sequences with the matched portion to the adapter sequence indicated in lower case are listed below. The sequence names are their ZMW IDs. The matched sequences (lower case) are normally not perfectly matched to the adapter sequence. In addition, there are some long regions without a good match indicating the quality was too poor for the adapter to be matched. For example, in the first sequence (ZMW ID 7), the first two segments (spilt by the adapters) are quite long comparing to later ones. In this sequence, the subread should be about 164 base pair long.

>ZMW ID 7 (SEQ ID NO: 14) GGCCGCTCTGTCCAGCGATTCGCCGTGTTACCGTAATCGCTCAAGGCAGCCCTCA CGCTTCAGCGCGGTGTCTGTAGGATAGATCTTTCCGAGCGACAGAGTGGACGGC CCTCGAAGAGGACTGGCCCGGCCTCGAGCCTGAGATCTGCGTTAATGGCTCCCG ATAGAGTCCGTCGGCTAGTGGTTGGAGCTCTCGCGCGCTCCTAATAACTCGCTGC GCTTCCTCGCAGCAGCAATCTACGCGTCCACTCTTCAGCTCAGACTAACAACCTC GCGAAGACGGAAGGAGAAGAGGCAGTATAGGGATGAGGTCATCGCGAAGGCCG CATCTATGCGCGAGGAAACCGGTGAGTAACACCGCGGGTGCATCCGTGTATTGT ATAGATCTCTGTGCCGAGCACCACAACAACGGAAGGGTCGCCGTTACGGGAAAA GCAGAAACGAGAACTCGGATAAACCTTTATCTTGGCTCATTCGCACGGCTCTCGG GACCTGCCTCTCGAGATAGAGGAATATGCGGTAGACGCGCTCGCGAAAGGACCT CAGCGGCATTCTTTTCTTACACATACAGCTCTTTTTATTCTGCGCCACGCCGACAG GTCTCCCCCAGATCCCTTCTTCAACCTAACCAGAGCTACAAGCTCTTGGCGGGGA AGGCGGCGGCGACGCGCATCTGTAGATACGCGGCGGCGGTGTATAGTTCTCCGA CGCGGTACGCGGTCACTCCTGGCATGCTCGCAAGGGTGTAACTTAGATAGCTCC GGGTTTCCCCCGACTTCCCCAGGCCTGGCAGTAGGAGTAGGCGTCCTTTGCGTTA GATTCTTCGTTTCTGCCTACAAACAAACCACAAACACGCCAGATCGAGGAATGT GAGGAACCACGAGCCGCAAGAACTCCATCCGCACGCGCCTACACCCGAGTACTA TTTGGTTCGGGGCGTGGGTGACCCATTCCACCGGCCTGTGATCGACAGGACCCCT ATAGGCATCTATACTCTCGGAGCCTGGATATCGTACGGTGGCTTTGGCGGGGGTC GGACCGGCATATATCTCTCCATGCGTCATCTTAGAGCACAGGCAGTATTTCGGTA CACAGAAAAAGGACGAGACAGGACGAGTCGTCCTGTGCAATTCTGGCTCGTAGC TCACACCGGTCAGTCCGCAGACTGCTCTCTCAACCAAACACGAGACGCAGGTAG GTTATGGCCAAAAGGAAGACCGAGGATTTCAAACTCTCTGGCCGGAACCGCGTG GGACAGTTCACCTTTCGGCGCACGCCAATCTGGCAGTGCTATTGTCGCAGGCGCC CGGGGTTCATCTAAACGGATCGCTCGATATCTTAAATCCTCGCGCTACAATGCCT TCCGGTGGAATGTAACTTCACCGTCCTTCTGGGGCCAGATAGCCCCTCACCGCCA AGAACCAACCAACGAGGGAGGAGAAAGAACTGGACATTTACCCAGACCGTGTG GATGTGCATCCGCGACCGGCTTAGATGGTCCTCAAGGCTGAGCCTGGATTCCTGT GTCGGTGCTTAATCGCGCCGCTCACATTCCTTCTCGATATCTGGAGACAACAGGA CGGAGGTAGGAGGGAAAAGAGCGAGGGAAGGTCCCTCGCCAAGCACCCTATCA GGCAGTACCACAGGCCTTTCGCGACCCAACACTACTTCGGGCAAGACTTCTAAC GCAGTACCTTGAGTGACGGGGGCACGGTCCAAATCTCCCAGGCATTGAGCGGGT ATCCACGAAAAAGGACCCGGTCGTCCTGGCAATTTCCGGTGTCTCACCGGTTCCG CAGAatcttctctcaaacaacaacaacggaggaggaggaaatcggcaggagaAGACGTGCGTGTTTACACG GGTGTGTATTACACACCGGAATTGCCAGGACGGACCCTGGTCCTTGTCGGTGAGT GAATACCTTTCGGCGTCTACACACTCGTCACTCGAGCGAGAATCTAAACTAGGC AGAGGAAAGCGTAAGGAAGAGCTCTCCAAAAGCACCTTCCTGCACTCCGCAACG AACGTGCTCGCTTGTTGTCGCAGCTCCTGGGAACCACTCGCCGAAGGCCTTCGGT GGGTACTCTCTTAGGTCAGGTGTGTCGCGGTTGGGAGGATCCCCTCTCAAACATC CACATTTGAGGCGTTTTTTTAATTCACGGAAAAGGACCCGTCGGTTCCACCCAAA TTCCGGGTGTACTCACCGGTCCCCAGATTCTTCTATTCAACAAAAAACGAGAGGA ACCAACGGAGGAGGAGGAAAAGAGAGAAGATCTCGCAAGCACCCTAATCAGAG CAAGGGATACGGCGAGGAACCTACTTGGCCTTTCCGCGGCCGAACCCGTGGAGT TAACCCGAATTCAACACCTAGGACCTGGCGGCTAAGCAGTCTTGCGGGGCGCAT CGCCAGATACTACCACGCGCCTTGCAACGGTTCTCACGAAGGAGGACCCGGTCG GTCCTGGGCAATTCCGGTCGTACTCACGCCGAGTGCACGCGATACTCAATGCCGT CAACGCAACAAGCAGAACGGAGGCCAGGGACCGCCGTTTTGAGTTAGATGAGA CGAGGAATCTGCGGACCGGTGAGTACACCGCATAATTCGTGGGCCATGGATCGA CACGCTCAAGGCAAGCATCTGATTCGTGGAATGGATAAAAAGAAAACCTTCTTC CGCAACGCTCAACTGCCTGGCAGATTTGGCTGACGTTCAGGCCCCCAGCTCGCAC AGACACTGCCTTTTCGCGACGCGTACGTCTACCGAGTAGTCGTTGCGAGGCGTTC TTGGTCGGCCGAAGGCCCCAAACTCCAGGGTTGCTCGGTTGGAAGCCTGTTTTAT CCACCGAAGGAACCGCCGTCGGTCCTGCATGCTCCGTGATAGGCTCACGCGCTTT CCTCGGGCATGTATGGATCTTTCTCCATACACAAAGCAACAAGCGGAGAGGCAG GGAAAGAGAGAGCTAATCCCCGCAAGCACACCGCTATGCGGCAGTTGACGCAA GAACAGAGAGACAGCGGGCCTTCTCGCTTGGACGCCAATTCACACTCAGCCTCG GCTAGCAAGTCTTGCGGGGCACGCCACCATCTCAGGTGCTTGCATTTGAGCGGTC TGATTCCCACTGTATAGCGACCCGCGCTCGTCCTGGGCAATTCCGTGTACCCCAC CGGTTCCGCAGatctctctcaaccaacaacaaacggaggaggcaggggaaaagagagagatGCTGAGGCGGA AGCCGGTGAGTAGGCCACCGGAATTGCCAGGACGACGCCGGTCCTTTCGTGGAT AAAACCCGCTCAATGCCTGAAGTTCTGGGCGTGCCCGCAAGACTGCTAGCCGAG TTAGTGTTGTGGTCGCGAAATGGGAGGCCTGTGGTACTCGGCCTGATAGGGTGCT TGCGAGatctctctcaacaaacaacaacggaggagaggaggaaagagacggcaggatCCGCAAGCACCCCCTA CTCAGGCCAGGTACGCACAAGGCGCTGTTCGCCGAACGCCCCACACCTACTCCG GCTAGCAGTCTTGGCGGGGGGCAGCGCCCAAATCTCCAGGCATTGAGCGGGTTT AATGCCACGAAAGGACCGCCGGTCGTCCTGGCAATTCCGCGTGTACTCAGCCGG TTTCGCAGatctctcatcaacaacaagcaacgcgaggaggaggaaaaggagatgatCTGCGGACGCGTGAAG TACACCGGAATTGCCAGGACGACCGGTCCTTCCTCGTGGATAAACGCCCGGCTTC CAAATGCGCTGGCCAGATTTGGCGGCGATGGCCCGCAAGACTGCTAGCCGAGTT AGTGTTGGGTCGCGAAGGCCTTGTGGTACTAGCCGTGAGTAGGGTGCTTGCCGA GatctctctcccaaacaaccaacaacggaggaggaggaaaaagagagagatCCTCGGCAAGCACGCCTTATG CAGGCCAGTACCACGAAGGCCTTCGCGACGGCGGCAACAACTACTCGGCTACAA AGACTCTTGGGCGGGGGGGCACGGCCAAATCTCCAGGCATTGAGCGGGTTTATC CAACGAAAGGACGCGCGGTCGTCCTGGGCAATTCCGGTGTAGCTCACGGTTTCC GCCAGAatctgctctcaacaagcaacacggaggagggaggaaaagggggggaAAGAGAGATCTGCGGAA CCGGTGAGTACAGCCGGAATTGCCAGGACGCAACCGGGGGTCCTTTCGTGGATA AACCCGTCAATGCCTGGAAGAATTTGGGGGCGTGCCCCCGCAAGACTCGCTAGG CCGAGTAGCTGTTGGGCTGCGGGCGAAAGGCCTTGTGGTATCTCGCCTGATAGG CGTCGCCTTGGCGAGatctctgctcagcccaacagacagacggaggcagagaggaaaagagagagaATCCCT TCGCAAGCACGCCTATCAGGCCAGTACCACAAAGGCCTTTCGCGAGCGCGTCAA CACTAGCCTCGCTAAGCAGTCTTGGCGGGGGGCAGCCAAATCTCGCAGGCATGA GGCGGGTTTATCCACGAAAGGACCCGGTCGTCGCTGAGCAATTCCGGGTTAGCT CACCGGTTCCGCAGATCTCTCTCAACAACAACAAGCCACCAAACGGAGGAGGAG GAAAGAGAGAGATCTGGCGGAACGCGTGAGTACCG >ZMW ID 8 (SEQ ID NO: 15) GGTGGAGTACAAGCCACGGAATTGGCCACCGGGACGACGCACGCAGCACGACC CGGGTCCATTTCGTGGAATAACCCGCTCATGCCTGGAGATTTGGGCGTGCCCCCC ACCCCGCAAGACTGCTGCCGAGTAGTGTTGGTCGCCGAAAGCGCCTTGTGGCTA AGCCTGCCGCCCTGATCAAGCACGGGTGCTTGCGAGATTCCTCTCACAACAACA CCACGGATGAGGAGGCCAAAAGAGCAGAACTCTCGCCAGCACCCTACTTCAAGG CAGTACCACCAAGGCCTTCCGCGACAGCCCGCAACACCTACTCCGGCCTAGCCA GTCTTGAAGCGGGCGGGCAAGGCGCCCAACGATCCTCCAGGGCATTGGCCGGGT TTTATCCCACGAAAGGACTCCGGCGTGCCTGGCCCATTCCGTTGTACTCCACGCG GCTTCCGCAGCTCCTCTCCTCCAAACAACCAACCAAAAACGAAGAGGAAGGAGG CAAAAGAGAGAGATCATGCGGAACCAAGGTGAGTACAAACCAGAGAATATAAC ACAAGGACAGAACCAAAAAAGAAGAGAACCCATTCATAATCGATGATAACACA AACCGCTCCACAGACATATAAAGAAGAACGCACGAACACGCGGCGCGTCGCCA AACGCCAAGATAGCGAGTAAGCCAATAGATAAGAGAGCAAAACAAAGTCAGAC AGAGAAGACCATAATAGAGATAACAAAAAAAAAAAAAAAAAAACATAAAAGCT GATAAGAGAAAAAAAAGATGCTACAGAGAAAATATCATCTCCATCACACAACAC AACACAGAGAGGAAGGAAAGGAAAAGAAGAAGAGAAGATACATGCAGCACAC TAAATCAAGAGAAAAAAACCAAAAAAAAGACAAGATAACAAAAAAAAAAAAA AAAAAATACACACAAAAACACCAAACACACACAACAAACACACACCAAACAAC ACACAAACAAAAAAACAAACAAAAACACCAAAAAAAAAGAAAAAAAAAAAAA ACAAAAAAACAAGACACAAACAAACAACAAAAAACCAAAACAAAAAAACAAA AAAAAACAAAGGCCTTTCGCCAAAAGAACCACAACAACTACAAAACAGACTAG AACAGATACCATATAAGCGGAGGAGCAAAAGCACAAATACAAAAAATACCAGG CATATTGAAAGACAAAGGCGATAATAATAAACCACGAAAGGACCGGTCGTAAA ACCTGGCAATTTCCGGCGTGTACTCACCGTTCCGCAGatctctcctcacccaacacaaccggacgg caggaggcaaaagagagagaGATCTGCGGAACCGCGCGTACACCGGAATTGCCAGCCGGA CGACCGGCGTCCTTTTCGTGGACTACACCCAGCTCAATCCGCCTCTGGAGATTTG GGCGTGCCCCCCGCCAAGGCCGGACGGACCACTGCTAGCCGAGTCAGTGTGATG GGGCGCCTCTGGCCCTCCGGCCCTTTGGCGGGGCGGGTTTGCCTTCCGACCGTGG ACGGGTCGCCGAAAGGCCGCCGTGTGCTCGGTCACTCCGCCGCCTGAAATAGGC GCTGGGCTTGGGGAGATCTTCTCCTCAACGCGTCCGTCTGGCAATTCGGGTGGGC GCCCCGGGAGCGGGAGTGACGCGCAGGAAAGAGAGAGCGCTCTGCATGCCGCC CCTATTCCCCAGGCGAGGGCGCGACAGAGAAGGGCCGCTGTGTTCTGCTGCGGC CACGAGCATACTGCGGCCTATGTAGTCGTGGCGGGGCGCCCAGATCTCCCAGGC ATTGAGCGGGTTATCCACGAAGCTTATCTCCCGTCGTGGCCTTGGCCAACGCCCT TCCGGTGTACTCATCTGGGTGACGGCGATCTCGCGCCACGCCATTATAAGAGCG GCAGGAGGGAGACGCGCCGAGAGCATGCTGCTGGAACCGCTGAGCGCGTTAAC AGCCGGAGTTTTCTGTGCCTAGGACGGGCTGTCGAGACCGTGGTCCTTTGTCGTC GCTACATACCCGCTCAATGCCTTCGGAGATTGGTGGGCGTCTGCCGGCCCGCGA AGGCACGGGCCTCTCCGGAGGTAAGCCGCTGTGGTGGGATTCGCGAAAGGGCCT TGTGGTACTGGCCTGATAGCGCGTTTCCGCGCTTGCGCGAGCGATCTCGTCTGCG AACATAACCAAAACGGGGAGGCGGCGGCGGAACAGAGAGAGCAGAGTCCTGCG CGCCCCCCTCTCACCCGGTCGCGGCGCGGCGATCGATGCACCACAGGCGCCGCT TTCGCGGCCCAACATCTCACTACTGCGCGCTAGCGCTCTGTGCGGCGGCTATACT GTCCAAGATGCGTCCTACCGGGCAGGCCGCCGCCCGGCACCAGTCGCAGCATCC TGGAGCCCGCGGGTTTCAGTCCACGGCAGCAGGTGGACGCCCCCGGGCTCGTGG CCCTCGCGACTCTCCGGGTACGCACCCGGTTCCGGCAGGATCCCTCCATCAGCGC GGGCCGGGCGCCGGCCACAACAGACGGGGCCGCGGCAGGAAGGGCCGGGACCC AAGAAGAGAGAGATCTGCGGAACCGGTGAGTACACGGAATTGCCAGGACGACC GGGTCCTTCGTGGATAAACGCTCGCTTCAATGCCTGGAGATTTTGGGCGTGCCCC GAACTGCTAGCCGAGTAGTGTTGGGCTCGCGAAGCCCTTGTGGGTACTCCGCCTG ATAGGCGTGCCTTGCGAGatctctctcaacaacaagcaagcggaggaaggagggaaaagaaaggagatCGCT CCGGCAAGGCACCCTAATCAGGCAGTACCACGAGAGGGCCTTTCGCGACCAAGC ACTACTCGCGCTAGCAGTCTTTGCGGGGGCACGCCAAATCCTCCGAGAGGCATC TGAGGGCGGGTTTATTCCAACGAAAGGACCCGGTCGTCGCCTGGCAATTCCCCG GTGTAGATCACGCGTTTCGCGGGCAGAATtctctctcacaacgacagcaacggagagagcaaaagaaga gagatCGTGGCGGAACCGGTGAGTACACCCGGAATTGGCAGGAACGACCGGTCCTT TCGTGGATAAACCCCGTCCAATGCCGTCGGAGAATTTGGGCGTGCCCGCAAGAC TGCTTAGGCCGAGTAGTGTTGGTCGCCGAAAGGCCTTGTTTGTGACTCGCCTGAT AGGGTGCTTGCGGGGatgctctctccaaacaaggcacacggaggagggaggcaaaagagagagatCTTCGCA AGCCCGAGCCTATCAAGTGGCGCAGTACCCAACAAGGCTTCGGCGAGCCCACCA ACACTACTCGGGCTAGGCAGTCCTTGCGGGGCACGCCCAAATCCGGCAGCATTG AGGCGGGTTTTTTTCTTTTTTAAAATCCAGGGTGCGGCTAAAGGACCCGGTCGTC CTGGCAATCCGTGTGTACCTCCCGGTCCGCAGatctgctccaaacagacaacaacgggaggcagagg aaaagagagagatCTGCGGAACGTCGTGTGAGTACGAACCGGAATTGCGCAGGACGAC CTGGTCCCTTCTTCGTGGATAGAACCCGCCTCAATGCACTGGAGATTTGGGGCGT GGCCCGCCGCAAAGACTCCGGCTTAGCCGAGTAGATGGTTGGGTCGCGGATGCG CGAAAGGCCTTGTGGTACCTCGCGTTTTTTTTTTTTATTTGTTCTTCCAA >ZMW ID 19 (SEQ ID NO: 16) CTTGTTGGGTCGCGCAACAGTGGGCCTGTGGTAACTGAGTTTTGTTCAGGCCTGC ATAGGTTGTGCTGCGAGTCTCTCTCGTGAAGCAGAGACAGACGGGAGGCGGAGG AAAAGAGACGCCGGATATGATCCGAAGTTGTTATCTGCAGCACCTATCGGCAGT ACCACAGTGCCTTTCGCGACCCATAGCACTACTCGGCTAGCCAGTTCTGCGGGGG CACGCCAAATCTCAGGCATTGAGCGGGTTATCCACGAAGACGGAACCCGGCGTC TGGCAATCGGTGCTACTCCGGTTCGCAGCATCTTCTCACACAAACAACGGGGGA GACAGGAAAGAGAGAAAGATCAATGCGAACCGGTGAGTCACACCAGGATTCGC CAGGCGTACCGGGTCCCTTTCGTGGATAAACCCAGCTCAATGCCTGGAGATTTTG GCGTGCCACCGCCAGACCTGCTCAGCCGAGTATGTTGGGTCGGAAAGGCCATTG TGGTACTAGCCTGATAGGGGTGCTGTGCGAGATCTCTCTTCAACACACAACGCAG CGAGAACGGTTAAGGTAAACGGAGAGTTCTCGGAAGCACCCTATCGGGGGCAAG TCCACAGGAGGCCCTTTTCGCGACCCCATGACACTACTCGGGGGGGTCTTCGCAG TCTTAGCCGGGGGCCGCCCAAATCTCTCAGGCATTTGGGCGGGTTTTTTTTATCC ACGAATGACCTGGCGGGCGGTCGTCTGGCAATGTCGGTGGTACTACACCGTTTCT CGCAGAGtctctctccaacatccacacaagcggaggaggaggaaaagagaagagatCTGGCGGAGCCCGGTG GTACTCGGAATTGCCAGGACGACCGGGGTCTTTCGTGATAAACCGCTCAATGCCT GGAAATTTGGGCGTGCCCCCGCAAGACTGCTAGCCGAGTAGTGTTGGCGGTCGC GAATGGCTTGTGTACTGGCCTGAATAGGGTTGCTTGCGGACGatcttcgtctcgaaacaacaa caaacggaggagggagggaaaagagagagatCGTCGCAGCACCCTATCAGCCGCAGCTACCACA AGCCTTTCGCGACGGGCAACGACTACTTTGCGGGGAGCAGTCGTTGCGGGCCAC GCCAATCTCGCCCAGGCATATTCGAGGCGGGTTTTATCCCGCGGAGGGAGCCCG GTCGGTCTGGCAATTCGGTGTACTCGCACGGTTTCGCAGATCTCCTTCTCAAGCA ACAGGGGGGGGGGAACAGAGGGGGAGGGAGGCAGGACCAAGAGGAGGATGAT CCTGCGGAAACCGGTGAGTACAGCCGGGACATTGCCCAGGACGACCGCGCAGCC GGCCGCACCGCCCCCCCGGTGCGGTCCTTCTCGTGGCCGCAGACGCCCCGCCCAC CGGCGCCCGTCAGTCCGCCCGTGCCGGAGAAGTATTGGGATGGGCGTGCCGCCC GCAAGACGTGCTCACGCCGAAGTAGTTGTGTTGGTGGCGCTGGAGGGTACTTGT CGGCGAAAGGCGCTTTCGGGTAGCTGCCTGATAGGCCGTGCTTGGAGatctcctctcaac aacaacaacggaggccacggaggcaaagagagctagatGCTCGCAGCGACTATCCCGGCGCAAGGGC CTCATGTATGGAGCCGAACACTCAGCTCGGGCCGCTAAGGCGGCTCTGGCGGGG CCGACGCCTCGCGCGCGCTCGAGGCTCGGGTTTATCCGCACCGACGGTACGCCG GTCGTCCTGGCATCGGTGTCACCTCACCGTTCCGCAGATCTCTCGCTGCCGACCA AGCAAGCCAACCGGGGAGGCCGGGGAAAAGATGATCGAGATCGTGCGGACGCC TGGTGACGTACACCGGATTGCCAGGGACTACGACCTCCCTTTCCCGGGCTCCTCT TCGTGGTATCAAGACCAGCAACGAAACCAGAGCGCTCACATGGCCTGGACGGGT TTGCGCGTGTCCGGCAAGACTGCTAGGCGCGAGATAGGTGTTGGGCGTGCGCGA AGGAAACCTTAGTGGTACTAAGAAGCCTGATAGGGCGTGCCTTAGCGAGATCTC TCGTGCACAGAGATTTTACTTCGCCCACCACAAACAACCGGAAGAAGGACGGCC AACAGAGACGAGATCCTCTCGCAAGCACCCCTATCAGGCAGTATAGCGCACAAG GCCTTTCGCGACCCAGCACTACTCGGGTCGCTCGGCAGAGTCTTTGGGGCGCGCC AAATGTGCCAGGCATTGGACGGCGTTATCCCCGAAAGGGACACCACGGTCGTCC TGCAGAAGCGTGCCGGTGTCACTGCACCGGTTCCGCGCAGTCTCtcttcgctcaacaagcag acaacggaagcggaggaaaagagagtagatCTGGCACCGGGTGAGTACTACGCAATTTTGCGCCA GGCAGCACGGGTCCCTTCGTGGATAGAACCCGGCTCATGCCTGGGACTTTGGGC GTCGGCCCCCGCAAGACTGCTAGGCCCGAGTAGTGTTGGGTCGCGAAAATGGCC TTGTGGTACTACTCGCCTTAGGAGTACGCTTGTGAGATCTtctctcgcaacaaaccacgacgga ggcgggaggaaaagagagagaATCGGTCGCAAAGCCCCACTACATCAGGCAGTACCCTACA AGGGCCTTTCGCGACTCCAACACTACTTCGGCTCTACGTCAGTCTTGCGCGGGGG CAGGGCCGAATCTCAAGACATTGACGCGGGGTTTCTCCACGGAGGACGAGATCC GTTCCTTGTGCAATTCCGTGTACTACAGCCGGTTTCGCAGATCCTCTCCCAACAA GCAACGCGAGGCGGCAACGGAACATGAGAGAGATCTGGCACCGTGAGTGTACG CACGGAATTGCAGGCACGACGGGTCTTTCGTGGATAGTCAACCCGCTATTGCTGG AGATTTGTGCGTTGCACCCAGCAATGACTGCTAGCGGCCGACGTACGACGGGGT TAGGAAAAAGGGGTCGCGAAGGCCTTTGTGGTAACTACCGGCTGATAGGCGTGC TTGGCGAGATCCTGCTCTCCTCTCGCACTAACAACAGCGGGGAGGCCTGGAAGA GGAGAATTCTTCGCCAGCCGCCCGATCCAGACAGCATAGTACTACACCCGGTGG CTTCTTCGCGCCCACACTACTCGGCTCGACGATCTTGCGGGGCACGCCCAAAATC GTCCGCAGGGCCTTGAGGCGGGTTATCCACGTAAAGGCCACGACCGGTCGTCCT GGCGACATATCTCGGTGTACTCCGCGAGTTCCGCTCGATCTCTTCTCGATATCAC CAACGTGAGGCCAGGCGGCAAAAAGAGAGAGTCTGCGAACGCGGCTGACGATA CACCGGATTGCAGGACGACCGGGTCTTTATCCGTGGATAGACACCCGCCATGCC TGGAGATTTGGCGCGTTGCCCGCAAGACTGCTAGCGAGTAGCTCGTTGGGCGTC GGCCGAACGGCCTTGTGGTACTGGCTGATAAGGGGTGCTTGCGACGatctcttccttcaca acaacaaccggaggaggaggaaaagagaggaAGGATCTCGCAGCACCCCTACCTCAGGCAAGTA CCACAAGGCTTTCGGACCCAACCTACCTCGCTAGCAGGTCTTGCGGGGGCCACG CCAAATCTCCCCAGGCATTGAGCAGGCGTTTATCCAACCGACAAGCCTCGCCCG GGCGGCGCCCGCCCGCCCAGCCTGTCTCCTCTTCTCTTTCTCTTTCTTCTGGCGCT CGCCTCCTCGTCGGTCCCCGGCGTTCCGGCCCGGCGTCCCCTCATGTCTCGCCGC GCGCCCCCCTCCTCCTTTGCCTGCCCGCTCTCGCCCCCTGTTTCCTTCCACGCTGG CTCGCGCGTGCGCTGTCACTCCCGCCCTCCCGGTCCGCAGA >ZMW ID 21 (SEQ ID NO: 17) CCCGCAGACTGCTAGCCGAGTAGTGTTGGGTCGCGAAGGGCCTTGTGGTACTCTC CGCCTGATGGGGTGGCTTGCGAGAACGCCCCCGCGCCAAAAAAACATGCTCTCC TCCACAACAACAACGGAGGAGGGTGCTGCTTTAGGAAAAGAGAGAGATTCGCA GCCCCACGCAGCCCTAGTCCCGCAGCAGCGTACCCACCCACCCCAGCGCCTGTTC GCCGACCGCCACACTACCGGCTTAGCAAGTCTTGCGGGGCACGCCCAAATCTCC CGGGCATTGAGCGCGTTTTACTCCACCGGAAAGACCAGACCTCGGGCGTCTGGG CATTCGGTTGCTAACTGCACCGGTTTTCCCCGCAGatcttttctcacacaaccacggggcggaggaa aagagagagatCTGGCGGTGAACCGGCTTGGTACACCCGGAATTGGCCCAGGGACGAC CCCGGGTCCCTTTCTCGTGGATAGAACCCGCCTCCATGCCTGGAGATTTGGGCGT CCCCCCGCCAGACTGCTAGCCGAGGTAGCTGTTTGGGCTCCGCGAAGGGCTTTGT GGTACTGCTGAATAGGGTGCTTGCGAGATCTCCGtctccaacaacaacaacaacggaggaggagga aacatgaagagagatCCTTCGCAAGCACCCCTAGTCCAGCGGCAGTACCAACAAGGCCTT TCGGCGACCCAACACGTTACTCGGCTAGCAGTCCTTGCGGGGGCACGCCCAAAT CTCCCAGGCATTTGAGCCGACGCGCGTTTTTTTTATGCCCACCGAAAGGGGACCC GGCCGTCCTGTGCCAAATTCCCGGTGTACTGCCACCCGGTTCCGCAGATtcgtctctcca acaacaacaacggaggaggaagggaaaagagagagatCTGCGGACCCGGTGAAGCTCACCGGAAAT TGCCAAAAGGAGACCCGGGTCCTTTTTTCGTTTGGATAAACTCCGCTCATGCCTG GAGATTTGGGCGCGTGCCCGCCCCGCAAGACTGCTTAACTAGCCGAGTAGTGTT GGGTCGGCGAAAGGCCTTGGTGGTAACTGCCTGATAGGGTGGGCGTTGGCGAGat ctcctttcaacaacaacaacgggagggaggaggaaaagagagagatCTCGCAAGCAAGCCCTATCAGGCGT ACCACACGGCCTTTTCGCGGAACCAAACACCTACTCCGGCTAGCAAGCTTCCTGC GGGGGGCCACGGCCAATCTCCAGCCATTTGAGCGGGTTTTTATCACACGAAGAC CCGGCCGGTCTGGCAATCTCCGGTGTAGCTGCAACGCGGTTCCGCAGatctcttgctcaac aacaacaacggaggaggcaaaggaaacagagagagatCTGCGGAACCGGTGAGTCACCGGAAATTT GCCCAGGACGACACGGGTCCTTTCGTGGATAACACCGCCAATGCCGTGGGAGAT TTGGGCGTGCCCCGCAAGAAACTCTGCCTAGCCGAGTACGTGTTTGGGTCCGGC GAAAGGGCCTTGTGGTAATTCGCCTGATAGGGTGCTTGGCGGAGCatctctctcaacaacg gaaaaacggaggaggagggaaagagaggagatCCTCGCAAAGCACCCTATCAGGCAGTGACAAC AAGGCCTTTCGCGACCTAACACTACTTCGGCGTTAGCATCTTTGCCGGGGGCAGG CCCAAATCTCATACAGGCATTGGAGGCGCGGGTTTTATCCACCCGAAAAGACCC GCCGGTCTGGCGGGGCAATTCCGGTGGTACTTCAACGGTTTCCCGCCAAGAatttctc ctcaaacaacaacaacggggaggaggaaaagagagagatCC >ZMW ID 25 (SEQ ID NO: 18) CCCGTCTGAGCCCGGCGTTCCTATCCACGACCCCGGACCCCCGCGCCTCGTCCCC TGCGCCGCAACTGTCCGGCCCTGCTCAACCCTCCGGTTCCGGCCAGatctcctctaacaac accaacggaaggaggaggaaaagagatgacgatCTGCGGAACCGGTTGAGTACACCGGAATTGCC AGGACGACCGGGTCCTTTCGTGGATAAACCCGCTCATGCCCGGAGAATTTGGGC GGTGCCCACGCAAGACCTCGCTCCGATTCAGCGATAGTCGTTGGGTCGCGAAAG GCCTTTGTGGTACTGCCTGATAAGGGTGCCTGCGAGATCTTCTCAACCAGCACAA GCGGCAGGGAGGCGAGGAAAAGAGAGAAGATCTCGCACGCACCCCCGCCTATC AGCGCAGTACCACAAGGCCTTTCGCGACCAACAACCTACTCGGCCCGCCTAGCA GTCTTGCCGGGGGCACGCAAATCTCCAGGCATGAGCGGGTTATCCACCGACAGG ACCGTCGCGTCGTCCTGGCAATTCCGGTGTACTGCACAAGGCTTCCGCAGGCATC TCTCTCAACCCACACCGCAACGAGGAGGAGGAAATACAGAGAGAGATCTGCGG AACCGGTGAGTACACCCGGATTGCAGGACACCGGGTCCTTTCCGTGGATAACCC GTCGAATGCCCCGGAGACTTTGGGCGTGCCACGCAAGATGCTCAGCCCGAGTAG TGTCTGGGTCGCGAAAGGCCTTGTGTACTGCTGATAGGGTGCTTGCGAAGatctctctc aacaacaacaacggggaggaggaaagagatgagatCTCGCAAGCAACCCCTATCAGGGCAGGTCAC CACAAGGGCCTCGTATCGCGACCCACACTACTCGGCCTAGCAGTCTTGCGGGGG GGCACGCCGAAATCTCCAGGCATGTGAGCGGGTTTATCCCGCGAAAGGGCCACG CGGCTCGTGCTGGCCGAATTCCGGTGTACACTCACCGGTTCCGCAGATCTCTTCT CCATCAGCACAACAACGAGGAGGAGGAAAAGAGCAGGAAGATCTGCGGAACCG GTGACCGTACACCGGATTGCCAGGACGACCAGGGTCCTTCTCGTGGATATACCC GCTCAATGCCCTCGGAGATTTTTGGCCGTGCCCACGCAAGAATGCTAGCCGAGT ATTGTTTGGGTTCGCGAAAGGCCTTGTGGTCTGCGCCTGATAGGGTGCTTGCGAG tctctctcaacaacaacaccggagggaggacaagagagagatCTCGCAAGCACCCTATGCCAGGGCCGT ACCCCCACGGGGCGGGGCCTGGTTCGCGAGCCCAAACACCTACTCGGCTAGGCA GGTCTTGCGGGGCACGCCCAAATCTCCAGGCATTGAGCGGGTTTATCACGACAG GACCCGCGTCGTCCTGGCATTCCGTGTGTACTCCAACCGGTTTCCCGCAGatctatctca acaacacaacggaggaaggtaaggaacagaggagagatCTGAGGAGAAACGCCGCGTGGAGTACACG GATTGCCAGGACGGACCGGGTCCTTTCGTGGATAAACCCGCTCAAATCCGGAGA TTTGGGGCGTCGGCCCACCGCAGACTGCTAGCCGAGTACTGTTGGGTCGCGAAA GGCCTTGTGGGTACTGCCTATAGGGTGGCTGCCGAGatcttctctcaacacacacggagggcagcg aggaaaagagagaCAGCTCTCGGAACGCCCCTATTCAGGGGCCAAGGCCTTCCCGCTCG GCGACCCCACACTACTCGGATAGCCAGTCTTGCGGGGCCACGCCCAAAATCTCC AGCCATTCGGAGCGGGTTTAATCCACGAAAGGACCCCGGTCGTCCTGCAATTCC GGTGTACTCACCGGTTCCGCAGATtctctctcaacaacaacaaccgagaggagggacggaaaagagagacg atCTGCGGAACCGGTGAGGCTACAGCCCGGAATTGCCAGGACGACCGGTCCTTCT CGTGATACAACCCGCCTCAATGCCGAAGAATTTGGGCGTTGCCCACGCAACGAC TCGCTAGCCCGACGTAGTGTTGGGTCCGACGAAAGGCTTGTTTGCGTACTGCTGT AGGGTGCTTTGCGAGATCTtcgctctcacaacaacaacggaggcaggaagggaaaagagagagTCCTCGC AAGCACCGCCTAGTCAGGCAGTACCACAAGGCCTTTCCGCGACCGCAACAACTA TCGGGCCGCTAGCAGTCTTGCGGGGCACGCCCAATTCTCCAGGCTTGAGCGGGTT TTATCACCGAAGGACCCGGTCGTCCTGGCAATTCCGGTTGTACGCTCACCGGTCC GCAGatctcctctcacacacacaacggaggaggaggaaagacgagagatCTGCGGAACCGGGGTGAGTAC ACGGACATTGCCAGGACGCCGGGTTCTTTCGTGGATAAACCGCTCAATGCCCGG AGATTTGGGCGTGCCCCACGCAATGACTGCTAGCCAGTAGTGTTGGGCTCGCGA AAGGCCCTTGTGGGTACTGCGCCTGATAAGGGTGCTTGCGAGatcttctcaacaacacaacga gaggagggaaaagagagagatCTCGCAAGCACCTATCAGGCGTACACAACGGCCTTCAATC AGAAAAAAGACCCAGACACTACTCGGCTAGACAGTCTTGCGGGGGCACGCCCCA AATCTCAGGCATTGAACGGGTTTATCCACGAAAGGACCGCGGTCGTCCCCTGGC AATCTCGGTGTACTCCAGCGGTTTCCGCACAGATCCTCTCCTCCCACACGCCcaaca acaacaacgcgaggaggaggcgaaaagacgagagatTCTGCGAACCGGTGAGTACACCGGAATTGC CAGGCCGGACCGGGTCCTTTCGTGGCTAAACCCCGCTCAATGCCGCGGAGATTT GGGGCGTTCGCCACGCACGACTCGCTAGCCGAGTAGTGTTGGGCTCCGCCCCGG AAAGGCCTTGGTGGCTACTGCCTGATAGGGTGCTGCGAGatctctctcaagcaacaacaacgga ggacgggaggaaaagacgacgacgcatCTCGCCACGCAGCCCTAATCAGGGGGCAGTCACCGG GCACAAGGCGCTTTCGCGCACCCCATCACACTCAATGCGCGCCTGGAGCAGTCC ACCCGCTTGCCGGGAGGCCTCGGCACGGCCAAACGCGCCAGATCTCGCACGGCA TCCGTGGAGCCGCGGTTTACTCCCACGAAGAGGATCCCCGGTCGTGCGCTGGGC AATTCCGGTGTACCTCGCTTGCAGCCCGGCTTCCGCATGATCTCCTCTCCAATCA ACAACAACGGAGGAGGGAGGAAAAAACGAGGAGCAGATCCGTGGCGGACAAC GCCGGTGAGGTACAACCCGGGAAATTCCGCCGAAGCAAACGGCGACCGGTCTCT TCCCCACGCAACACCACGCGATCAATCCAAACAAAAAAAAAAAAAAAAAAAAA AAACGTGGAAACCAAGAGGAACACCACCCGCCCCCCGGGCACCC

Example 5 Run the Polling Algorithm to Generate One Consensus

From the initial 179001 sequences in the accepted set, the first poll produced the first base of the consensus that is C with about ⅔ of the bases (See Table 4). The distribution of the base were 114994, 26000, 19299, 18708 for C, A, G, T respectively. For the first two iterations, we skipped the scoring sequences from the rejected set simply because there is no chance for those sequences to be scored high and returned to the accepted set. The Overlap Matching alignment was used for the scoring starting at the third step. The cutoff was 0.25 per base. A sequence with a matching score less than the cutoff would be returned to the accepted set.

We started at the third step with 82079 in the accepted set and 96922 in the rejected set and the consensus sequence is CT. The polling found G is the next dominant base and rejected 44131 sequences. Then the Overlap Matching alignment of the 141035 sequences (96922 plus 44131) to the consensus sequence CTG and found 14915 sequences with good matches, and returned them to the accepted set. Therefore, at the end of the third step, we have consensus CTG, 52863 in the accepted set and 126138 in the rejected set. The data points mentioned in the paragraph are highlighted in Table 4.

To illustrate the process furthermore, nine subreads at step 5 are listed below. We started with the consensus CTGC. The next bases to be polled are lined up. It is obvious that G was the dominant base and should be incorporated in the consensus. Sequence 4 should be moved to the rejected set. Notice that sequences 3, 4, and 5 have an extra G at the fourth position (can be considered as an insertion there). They must have been rejected initially in step 4 and then returned back into the accepted set because they had high enough matching score in the pairwise-alignment analysis.

(SEQ ID NO: 19) 1 CTGC G (SEQ ID NO:19) 2 CTGC G (SEQ ID NO: 20) 3 CTGGC G (SEQ ID NO: 21) 4 CTGGC A (SEQ ID NO: 20) 5 CTGGC G (SEQ ID NO: 19) 6 CTGC G (SEQ ID NO: 19) 7 CTGC G (SEQ ID NO: 19) 8 CTGC G (SEQ ID NO: 19) 9 CTGC G

The consensus generation was set to be terminated if the number of early-terminated subreads more than doubled from one step to the next. This round of consensus generation stopped when the terminated subreads jumped from 2934 to 27713, a more than nine fold increase. The generated consensus sequence exactly matched the amplicon sequence, representing 44490 sequences.

TABLE 4 The statistics at each step of the polling process for the first consensus SEQ ID Consensus generated Accepted Rejected Newly Newly Step NO: (omitted after 30) size Size rejected returned 1 — C 179001 0 64007 0 2 — CT 114994 64007 32915 0 3 — CTG 82079 96922 44131 14915 4 22 CTGC 52863 126138 9965 12798 5 19 CTGCG 55696 123305 11862 53929 6 23 CTGCGG 97763 81238 49577 17871 7 24 CTGCGGA 66057 112944 17367 10949 8 25 CTGCGGAA 59639 119362 11308 13704 9 26 CTGCGGAAC 62035 116966 21156 12550 10 27 CTGCGGAACC 53429 125572 11945 34538 11 28 CTGCGGAACCG 76022 102979 34638 23424 12 29 CTGCGGAACCGG 64808 114193 20730 13214 13 30 CTGCGGAACCGGT 57292 121709 14288 12986 14 31 CTGCGGAACCGGTG 55990 123011 9236 11890 15 32 CTGCGGAACCGGTGA 58644 120357 12587 14177 16 33 CTGCGGAACCGGTGA 60234 118767 14136 15506 G 17 34 CTGCGGAACCGGTGA 61604 117397 17413 11500 GT 18 35 CTGCGGAACCGGTGA 55691 123310 10119 13459 GTA 19 36 CTGCGGAACCGGTGA 59031 119970 15801 16710 GTAC 20 37 CTGCGGAACCGGTGA 59940 119061 16114 33422 GTACA 21 38 CTGCGGAACCGGTGA 77248 101753 34352 14127 GTACAC 22 39 CTGCGGAACCGGTGA 57023 121978 15523 16687 GTACACC 23 40 CTGCGGAACCGGTGA 58187 120814 26073 16440 GTACACCG 24 41 CTGCGGAACCGGTGA 48554 130447 13237 13830 GTACACCGG 25 42 CTGCGGAACCGGTGA 49147 129854 11748 14166 GTACACCGGA 26 43 CTGCGGAACCGGTGA 51565 127436 8564 17263 GTACACCGGAA 27 44 CTGCGGAACCGGTGA 60264 118737 21062 10416 GTACACCGGAAT 28 45 CTGCGGAACCGGTGA 49618 129383 8836 10108 GTACACCGGAATT 29 46 CTGCGGAACCGGTGA 50890 128111 11249 13467 GTACACCGGAATTG 30 47 CTGCGGAACCGGTGA 53108 125893 9505 21651 GTACACCGGAATTGC 31 65254 113747 18871 20863 32 67246 111755 22646 19167 33 63767 115234 18193 15587 34 61161 117840 13398 17384 35 65147 113854 20073 8978 36 54052 124949 11515 14100 37 56637 122364 17025 16122 38 55734 123267 16778 14578 39 53534 125467 13981 12212 40 51765 127236 11483 26561 41 66843 112158 35648 12012 42 43207 135794 6848 12433 43 48792 130209 12659 16392 44 52527 126474 15338 6996 45 44190 134811 8778 15930 46 51347 127654 12923 17754 47 56186 122815 14998 17921 48 59124 119877 16010 14241 49 57376 121625 10879 11652 50 58174 120827 13281 9551 51 54473 124528 14658 13924 52 53772 125229 14505 13995 53 53299 125702 9755 9569 54 53167 125834 8883 9895 55 54236 124765 12433 7752 56 49618 129383 9334 19022 57 59375 119626 15454 10353 58 54349 124652 9130 14081 59 59385 119616 14028 6916 60 52362 126639 13466 10914 61 49905 129096 7646 18302 62 60669 118332 18664 11928 63 54055 124946 20405 14373 64 48154 130847 12198 17810 65 53920 125081 18203 13074 66 48957 130044 8908 8615 67 48836 130165 8893 18242 68 58362 120639 16139 17273 69 59678 119323 24104 9033 70 44794 134207 7642 13128 71 50470 128531 9146 19209 72 60729 118272 14745 13502 73 59694 119307 19389 13108 74 53637 125364 13656 9637 75 49854 129147 6114 16206 76 60196 118805 15221 9720 77 54953 124048 11659 15499 78 59056 119945 16262 9596 79 52660 126341 12704 10582 80 50813 128188 6209 13179 81 58065 120936 13201 10722 82 55882 123119 13667 4597 83 47116 131885 4400 10757 84 53786 125215 12359 10225 85 51981 127020 11935 7755 86 48141 130860 14030 20101 87 54561 124440 16701 12618 88 50845 128156 12717 9321 89 47830 131171 9451 11740 90 50505 128496 5981 19800 91 64728 114273 16319 12222 92 61067 117934 16241 6365 93 51659 127342 21032 5777 94 36893 142108 14631 9883 95 32651 146350 8044 23761 96 48925 130076 11346 15219 97 53376 125625 11708 22066 98 64327 114674 21640 12338 99 55638 123363 16424 10164 100 50030 128971 11730 13787 101 52759 126242 14268 7517 102 46720 132281 10893 11049 103 47602 131399 10905 16890 104 54333 124668 18417 10017 105 46699 132302 10524 13928 106 50890 128111 13712 12025 107 50000 129001 10652 10501 108 50659 128342 10203 15584 109 56869 122132 17881 7781 110 47635 131366 8276 9503 111 49747 129254 11847 11253 112 50053 128948 9315 6967 113 48696 130305 9998 9995 114 49695 129306 11436 7769 115 47036 131965 9204 7643 116 46554 132447 9071 8961 117 47535 131466 9866 6916 118 45681 133320 6519 16195 119 56465 122536 19096 9784 120 48284 130717 5765 9224 121 52886 126115 11413 9706 122 52359 126642 19003 2723 123 37299 141702 6838 15797 124 47494 131507 13657 18342 125 53432 125569 11954 7878 126 50637 128364 11854 13634 127 53716 125285 11775 9234 128 52499 126502 11018 11459 129 54308 124693 15627 8478 130 48544 130457 15301 4729 131 39378 139623 7013 16293 132 50146 128855 12080 12755 133 52331 126670 11221 16356 134 58998 120003 22818 10540 135 48357 130644 7920 10434 136 52526 126475 15722 10780 137 49256 129745 9569 5523 138 46896 132105 6868 9054 139 50775 128226 8095 9594 140 53980 125021 13734 4968 141 46947 132054 8485 7744 142 47956 131045 13700 14683 143 50705 128296 15142 8835 144 46203 132798 16167 13688 145 45583 133418 11996 9840 146 45346 133655 12349 16077 147 51023 127978 18671 10408 148 44751 134250 9391 8322 149 45692 133309 8604 12148 150 51259 127742 14748 6636 151 45187 133814 8758 5601 152 44099 134902 9162 12223 153 49242 129759 8446 9105 154 52073 126928 13358 7117 155 48035 130966 14938 3586 156 38904 140097 7751 9171 157 42595 136406 10721 11489 158 45649 133352 13279 7927 159 42607 136394 8931 10176 160 46189 132812 11630 7796 161 44705 134296 8667 7355 162 45762 133239 11408 12879 163 49629 129372 12977 9778 164 48918 130083 13895 9467

Example 6 Run the Polling Algorithm to Generate the Second Consensus

The remaining 131577 sequences in the rejected set from the last example were used as the input for the second consensus generation. The process stopped when terminated subreads increased from 2689 to 26488, almost ten times. This time the sequence generated matched perfectly to the reverse strand of the amplicon sequence, representing 41808 sequences.

TABLE 5 The statistics at each step of the polling process for the second consensus SEQ ID Consensus (omitted after Accepted Rejected Newly Newly Step NO: 30) size size rejected returned 1 — C 131577 0 49289 0 2 — CT 82288 49289 25463 0 3 — CTC 56825 74752 21285 17963 4 48 CTCG 53503 78074 15524 9754 5 49 CTCGC 47733 83844 7484 28820 6 50 CTCGCA 69069 62508 28852 15530 7 51 CTCGCAA 55747 75830 14982 11761 8 52 CTCGCAAG 52526 79051 16118 10449 9 53 CTCGCAAGC 46857 84720 10305 14724 10 54 CTCGCAAGCA 51276 80301 13134 24531 11 55 CTCGCAAGCAC 62673 68904 20412 8228 12 56 CTCGCAAGCACC 50489 81088 7678 15564 13 57 CTCGCAAGCACCC 58375 73202 19113 8873 14 58 CTCGCAAGCACCCT 48135 83442 15343 7349 15 59 CTCGCAAGCACCCTA 40141 91436 7052 15067 16 60 CTCGCAAGCACCCTA 48156 83421 12666 12094 T 17 61 CTCGCAAGCACCCTA 47584 83993 6985 16338 TC 18 62 CTCGCAAGCACCCTA 56937 74640 20854 12471 TCA 19 63 CTCGCAAGCACCCTA 48554 83023 8442 8653 TCAG 20 64 CTCGCAAGCACCCTA 48765 82812 10054 22863 TCAGG 21 65 CTCGCAAGCACCCTA 61574 70003 24213 11845 TCAGGC 22 66 CTCGCAAGCACCCTA 49206 82371 16506 10032 TCAGGCA 23 67 CTCGCAAGCACCCTA 42732 88845 8480 17900 TCAGGCAG 24 68 CTCGCAAGCACCCTA 52152 79425 14123 12569 TCAGGCAGT 25 69 CTCGCAAGCACCCTA 50598 80979 10021 13367 TCAGGCAGTA 26 70 CTCGCAAGCACCCTA 53944 77633 11707 12493 TCAGGCAGTAC 27 71 CTCGCAAGCACCCTA 54730 76847 13884 10290 TCAGGCAGTACC 28 72 CTCGCAAGCACCCTA 51136 80441 19186 9156 TCAGGCAGTACCA 29 73 CTCGCAAGCACCCTA 41106 90471 9797 14231 TCAGGCAGTACCAC 30 74 CTCGCAAGCACCCTA 45540 86037 13210 12774 TCAGGCAGTACCACA 31 45104 86473 10417 12477 32 47164 84413 13130 8108 33 42142 89435 6682 16091 34 51551 80026 13131 13281 35 51701 79876 11843 9937 36 49795 81782 14551 8109 37 43353 88224 4394 10992 38 49951 81626 9653 10094 39 50392 81185 14081 5273 40 41584 89993 9273 15104 41 47416 84161 12735 15665 42 50347 81230 13521 10807 43 47634 83943 12232 14538 44 49941 81636 14873 12238 45 47310 84267 7422 11357 46 51276 80301 17354 8468 47 42498 89079 13542 5153 48 34265 97312 7361 19444 49 46508 85069 14340 13229 50 45564 86013 10745 7477 51 42476 89101 7418 9868 52 45117 86460 10444 9509 53 44377 87200 9559 8617 54 43633 87944 10191 10655 55 44300 87277 10099 7424 56 41836 89741 8976 8308 57 41393 90184 11097 12270 58 42799 88778 7688 14412 59 49762 81815 16040 12832 60 46812 84765 14967 6667 61 38784 92793 6814 9283 62 41528 90049 8077 13523 63 47257 84320 11887 9707 64 45373 86204 11723 11109 65 45063 86514 10714 10190 66 44855 86722 10635 9505 67 44055 87522 8962 10352 68 45785 85792 13688 7877 69 40316 91261 6442 7949 70 42166 89411 10535 12419 71 44398 87179 10874 8866 72 42743 88834 9433 12484 73 46154 85423 6677 12519 74 52362 79215 7606 7689 75 52827 78750 7146 4760 76 50834 80743 14193 4157 77 41203 90374 14703 3249 78 30162 10141 57814 16293 79 39061 92516 11324 15240 80 43407 88170 10665 11497 81 44679 86898 10084 11059 82 46106 85471 6102 11146 83 51611 79966 16715 6762 84 42156 89421 12065 4884 85 35489 96088 3599 19131 86 51543 80034 12761 8347 87 47685 83892 14291 4150 88 38142 93435 7005 13226 89 44967 86610 10884 10223 90 44925 86652 6721 6796 91 45623 85954 8531 11722 92 49445 82132 13937 8084 93 44235 87342 7208 9343 94 47028 84549 6892 16241 95 57045 74532 17317 7852 96 48255 83322 11872 6496 97 43564 88013 8613 8032 98 43684 87893 6371 10758 99 48782 82795 9835 8802 100 48467 83110 9759 8202 101 47633 83944 10902 9744 102 47210 84367 11835 9750 103 45886 85691 13333 11132 104 44464 87113 7044 11864 105 50079 81498 12673 9357 106 47575 84002 10538 4318 107 42191 89386 5362 13386 108 51066 80511 8821 8202 109 51328 80249 19122 3497 110 36671 94906 7904 7765 111 37525 94052 7163 18501 112 49865 81712 10754 11545 113 51676 79901 16811 8088 114 44000 87577 8733 8088 115 44411 87166 11148 12028 116 46356 85221 9680 8129 117 45903 85674 5740 13360 118 54632 76945 13222 5479 119 48013 83564 13095 2910 120 38962 92615 5280 14109 121 48931 82646 11179 12921 122 51822 79755 12920 6550 123 46612 84965 8470 12408 124 51737 79840 16021 8535 125 45473 86104 16080 6694 126 37326 94251 9148 13840 127 43284 88293 12279 11998 128 44288 87289 7481 9448 129 47548 84029 16712 11273 130 43420 88157 9401 8686 131 44039 87538 10549 10398 132 45252 86325 11681 7452 133 42402 89175 13252 12448 134 43004 88573 8978 9704 135 45147 86430 7370 11084 136 50302 81275 12861 5168 137 44065 87512 7988 8605 138 46150 85427 10181 10613 139 48080 83497 12757 6971 140 43804 87773 11373 11609 141 45575 86002 11102 9298 142 45321 86256 10997 9291 143 45177 86400 11481 7816 144 43090 88487 8871 9371 145 45190 86387 9909 10369 146 47274 84303 7169 8117 147 49863 81714 11984 7030 148 46559 85018 8863 5573 149 44935 86642 11487 8142 150 43294 88283 9948 7906 151 42987 88590 10490 8716 152 42979 88598 12730 7147 153 39175 92402 8404 8161 154 40737 90840 9084 12699 155 46171 85406 12976 6695 156 41755 89822 9695 10354 157 44312 87265 11377 9028 158 43893 87684 6598 8552 159 47802 83775 15583 6781 160 40975 90602 7927 6816 161 41862 89715 13086 12534 162 43342 88235 9618 6643 163 42523 89054 11889 10580 164 43452 88125 9853 8209

Example 7 Run the Polling Algorithm on the First 100 Reads from the Data Set

This example took only the first 100 reads with 762 subreads from the data set as the input. The process stopped when terminated subreads changed from 13 to 134. The consensus generated matched perfectly to the amplicon sequence and representing 199 sequences.

TABLE 6 The statistics at each step of the polling process for the first consensus from 100 reads SEQ ID Consensus (omitted after Accepted Rejected Newly Newly Step NO: 30) size size rejected returned 1 — C 762 0 259 0 2 — CT 503 259 129 0 3 — CTG 374 388 180 59 4 22 CTGC 253 509 62 47 5 19 CTGCG 238 524 49 226 6 23 CTGCGG 415 347 212 102 7 24 CTGCGGA 305 457 78 40 8 25 CTGCGGAA 267 495 48 57 9 26 CTGCGGAAC 276 486 79 50 10 27 CTGCGGAACC 247 515 58 127 11 28 CTGCGGAACCG 316 446 126 99 12 29 CTGCGGAACCGG 289 473 101 70 13 30 CTGCGGAACCGGT 258 504 75 55 14 31 CTGCGGAACCGGTG 238 524 41 62 15 32 CTGCGGAACCGGTGA 259 503 52 64 16 33 CTGCGGAACCGGTGA 271 491 59 65 G 17 34 CTGCGGAACCGGTGA 277 485 76 52 GT 18 35 CTGCGGAACCGGTGA 253 509 48 52 GTA 19 36 CTGCGGAACCGGTGA 257 505 61 68 GTAC 20 37 CTGCGGAACCGGTGA 264 498 65 136 GTACA 21 38 CTGCGGAACCGGTGA 335 427 146 50 GTACAC 22 39 CTGCGGAACCGGTGA 239 523 71 72 GTACACC 23 40 CTGCGGAACCGGTGA 240 522 104 74 GTACACCG 24 41 CTGCGGAACCGGTGA 210 552 56 60 GTACACCGG 25 42 CTGCGGAACCGGTGA 214 548 49 62 GTACACCGGA 26 43 CTGCGGAACCGGTGA 227 535 29 79 GTACACCGGAA 27 44 CTGCGGAACCGGTGA 277 485 90 46 GTACACCGGAAT 28 45 CTGCGGAACCGGTGA 233 529 34 39 GTACACCGGAATT 29 46 CTGCGGAACCGGTGA 238 524 55 48 GTACACCGGAATTG 30 47 CTGCGGAACCGGTGA 231 531 44 93 GTACACCGGAATTGC 31 280 482 83 97 32 294 468 117 83 33 260 502 71 70 34 259 503 55 92 35 296 466 93 27 36 230 532 45 50 37 235 527 63 84 38 256 506 79 57 39 234 528 63 55 40 226 536 57 111 41 280 482 140 60 42 200 562 29 62 43 233 529 67 65 44 231 531 69 27 45 189 573 40 73 46 222 540 70 77 47 229 533 66 98 48 261 501 69 72 49 264 498 43 50 50 271 491 62 26 51 235 527 46 65 52 254 508 70 62 53 246 516 41 36 54 242 520 38 45 55 250 512 55 28 56 224 538 41 101 57 285 477 76 52 58 262 500 57 55 59 261 501 58 23 60 227 535 49 53 61 232 530 33 90 62 290 472 98 44 63 237 525 89 53 64 202 560 54 98 65 247 515 78 70 66 240 522 46 39 67 234 528 43 66 68 258 504 85 72 69 246 516 88 37 70 196 566 27 67 71 237 525 40 86 72 284 478 76 64 73 273 489 95 58 74 237 525 58 47 75 227 535 32 71 76 267 495 79 40 77 229 533 38 76 78 268 494 77 50 79 243 519 57 27 80 215 547 20 62 81 259 503 56 50 82 255 507 54 19 83 222 540 15 43 84 252 510 53 50 85 251 511 70 28 86 211 551 49 88 87 252 510 77 73 88 250 512 63 36 89 225 537 39 39 90 227 535 23 96 91 302 460 85 62 92 281 481 82 28 93 229 533 100 31 94 162 600 54 46 95 156 606 32 110 96 236 526 66 56 97 228 534 52 92 98 270 492 78 57 99 252 510 70 48 100 233 529 51 51 101 236 526 56 41 102 224 538 55 51 103 223 539 54 72 104 244 518 77 45 105 215 547 49 61 106 230 532 54 54 107 233 529 54 47 108 229 533 49 60 109 243 519 83 49 110 213 549 30 37 111 224 538 55 49 112 222 540 39 28 113 215 547 50 55 114 224 538 43 38 115 223 539 45 37 116 219 543 40 33 117 216 546 37 40 118 224 538 36 80 119 273 489 86 35 120 227 535 23 38 121 247 515 48 36 122 240 522 91 9 123 163 599 27 73 124 214 548 53 90 125 256 506 50 29 126 240 522 43 53 127 256 506 53 40 128 249 513 58 45 129 242 520 72 30 130 206 556 62 17 131 167 595 34 72 132 211 551 49 64 133 232 530 71 67 134 234 528 86 37 135 193 569 36 51 136 216 546 49 54 137 229 533 46 29 138 220 542 32 29 139 225 537 35 47 140 246 516 65 23 141 213 549 34 34 142 222 540 72 54 143 213 549 54 31 144 199 563 62 53 145 199 563 47 43 146 204 558 57 74 147 230 532 77 37 148 199 563 31 34 149 211 551 36 60 150 244 518 76 27 151 204 558 42 26 152 198 564 35 55 153 228 534 30 43 154 251 511 53 31 155 239 523 71 18 156 196 566 40 31 157 197 565 48 52 158 211 551 52 36 159 205 557 41 54 160 228 534 52 22 161 208 554 43 32 162 207 555 45 42 163 214 548 46 49 164 227 535 67 39

Example 8 Generating the Second Consensus from the First 100 Reads from the Data Set

This example started with 550 subreads left from example 6. The process stopped when terminated subreads changed from 9 to 102. The sequence generated matched perfectly to the reverse strand of the amplicon sequence, representing 174 sequences.

TABLE 7 The statistics at each step of the polling process for the second consensus from 100 reads SEQ ID Consensus (omitted after Accepted Rejected Newly Newly Step NO: 30) size size rejected returned 1 — C 550 0 203 0 2 — CT 347 203 99 0 3 — CTC 248 302 109 78 4 48 CTCG 217 333 56 45 5 49 CTCGC 206 344 36 132 6 50 CTCGCA 302 248 124 43 7 51 CTCGCAA 221 329 68 55 8 52 CTCGCAAG 208 342 66 36 9 53 CTCGCAAGC 178 372 36 66 10 54 CTCGCAAGCA 208 342 45 86 11 55 CTCGCAAGCAC 249 301 79 34 12 56 CTCGCAAGCACC 204 346 38 66 13 57 CTCGCAAGCACCC 232 318 81 42 14 58 CTCGCAAGCACCCT 193 357 57 36 15 59 CTCGCAAGCACCCTA 172 378 36 63 16 60 CTCGCAAGCACCCTA 199 351 54 39 T 17 61 CTCGCAAGCACCCTA 184 366 18 57 TC 18 62 CTCGCAAGCACCCTA 223 327 76 60 TCA 19 63 CTCGCAAGCACCCTA 207 343 30 32 TCAG 20 64 CTCGCAAGCACCCTA 209 341 41 94 TCAGG 21 65 CTCGCAAGCACCCTA 262 288 104 47 TCAGGC 22 66 CTCGCAAGCACCCTA 205 345 73 35 TCAGGCA 23 67 CTCGCAAGCACCCTA 167 383 31 60 TCAGGCAG 24 68 CTCGCAAGCACCCTA 196 354 44 59 TCAGGCAGT 25 69 CTCGCAAGCACCCTA 211 339 56 59 TCAGGCAGTA 26 70 CTCGCAAGCACCCTA 214 336 44 50 TCAGGCAGTAC 27 71 CTCGCAAGCACCCTA 220 330 68 53 TCAGGCAGTACC 28 72 CTCGCAAGCACCCTA 205 345 69 36 TCAGGCAGTACCA 29 73 CTCGCAAGCACCCTA 172 378 48 65 TCAGGCAGTACCAC 30 74 CTCGCAAGCACCCTA 189 361 53 44 TCAGGCAGTACCACA 31 180 370 51 52 32 181 369 45 36 33 172 378 29 77 34 220 330 57 56 35 219 331 65 40 36 194 356 56 22 37 160 390 8 49 38 201 349 45 32 39 188 362 51 21 40 158 392 30 68 41 196 354 51 66 42 211 339 55 39 43 195 355 45 64 44 214 336 60 39 45 193 357 36 36 46 194 356 69 34 47 161 389 50 23 48 136 414 37 69 49 170 380 55 53 50 170 380 43 41 51 170 380 37 37 52 172 378 39 36 53 171 379 39 36 54 170 380 33 51 55 190 360 43 29 56 178 372 44 31 57 167 383 40 42 58 171 379 29 64 59 208 342 69 57 60 198 352 59 41 61 182 368 34 32 62 182 368 36 41 63 189 361 54 40 64 177 373 48 57 65 188 362 52 50 66 188 362 46 27 67 171 379 36 43 68 180 370 53 31 69 160 390 28 30 70 164 386 41 57 71 182 368 55 37 72 166 384 28 56 73 196 354 34 65 74 229 321 36 25 75 220 330 35 20 76 207 343 53 18 77 174 376 64 14 78 126 424 32 56 79 152 398 35 71 80 190 360 41 41 81 192 358 39 44 82 199 351 25 42 83 218 332 80 24 84 164 386 33 20 85 153 397 22 97 86 230 320 70 20 87 182 368 46 17 88 155 395 22 63 89 198 352 48 41 90 193 357 31 35 91 199 351 48 43 92 196 354 54 20 93 164 386 23 44 94 187 363 39 65 95 215 335 62 35 96 190 360 45 29 97 176 374 44 38 98 172 378 27 40 99 187 363 38 50 100 201 349 37 38 101 204 346 47 40 102 199 351 52 46 103 195 355 49 51 104 199 351 35 51 105 217 333 52 36 106 203 347 45 16 107 176 374 11 57 108 224 326 40 30 109 216 334 72 12 110 159 391 33 34 111 163 387 26 70 112 210 340 38 41 113 216 334 55 36 114 200 350 54 34 115 183 367 40 45 116 191 359 43 38 117 189 361 26 47 118 213 337 63 27 119 180 370 45 13 120 152 398 15 74 121 215 335 49 43 122 213 337 52 23 123 188 362 32 50 124 210 340 74 34 125 174 376 56 32 126 154 396 38 74 127 194 356 53 38 128 183 367 24 31 129 194 356 50 51 130 199 351 37 46 131 212 338 53 28 132 191 359 53 25 133 167 383 39 52 134 185 365 36 47 135 201 349 37 33 136 202 348 45 24 137 186 364 33 34 138 192 358 54 43 139 186 364 41 18 140 168 382 31 50 141 192 358 42 36 142 191 359 52 38 143 182 368 48 25 144 164 386 27 38 145 180 370 45 53 146 193 357 23 27 147 203 347 53 46 148 202 348 35 20 149 193 357 58 29 150 170 380 33 25 151 168 382 27 41 152 188 362 54 25 153 165 385 35 26 154 162 388 34 52 155 186 364 46 36 156 182 368 48 41 157 181 369 45 26 158 168 382 23 44 159 195 355 69 23 160 155 395 28 24 161 157 393 37 64 162 190 360 42 25 163 179 371 50 35 164 171 379 34 37

The examples and embodiments described herein are for illustrative purposes only. Various modifications or changes thereof are apparent and are included within the spirit and purview of this application and scope of the appended claims. All publications, patents, patent applications, web sites, accession numbers and the like cited herein are hereby incorporated by reference in their entirety for all purposes. If different versions of any such citation are available, the most recent version at the effective filing date of the present application, the effective filing date meaning the filing date of the earliest priority application disclosing the sequence. Unless otherwise apparent from the context, any embodiment, aspect, step, feature, element or the like can be used in combination with any other. 

What is claimed is:
 1. A computer-implemented method of developing a consensus sequence from a plurality of sequencing reads of a nucleic acid target, comprising (a) receiving a population of raw target sequencing reads of a nucleic acid target comprising an anchor segment and an adjacent segment; the anchor segment being of known sequence and the adjacent segment being of unknown sequence; and at least some of the raw target sequencing reads containing sequencing errors; (b) evaluating the accuracy of sequencing of the anchor segment in different raw target sequencing reads by comparing raw target sequencing reads of the anchor segment with the known sequence of the anchor segment; (c) assigning a subset of the raw target sequencing reads into an accepted class based on reaching at least a threshold level of accuracy of the sequencing of the anchor segment; (d) polling nucleobases at a position equidistant to the anchor segment sequence in raw target sequencing reads in the accepted class to determine a consensus nucleobase, which consensus nucleobase is assigned as the first nucleobase of a nascent sequence of the adjacent segment; (e) assigning raw target sequencing reads having the consensus nucleobase determined in the prior polling step to remain in an accepted class and assigning raw target sequencing reads lacking the consensus nucleobase determined in the prior polling step to the rejected class; (f) optionally reassigning a raw target sequencing read from the rejected class to the accepted class by scoring similarity of the raw target sequencing read to the nascent sequence and reintroducing the raw target sequencing read if the sequence similarity reaches at least a threshold level of similarity; and (g) repeating steps (d), (e) and optionally (f), except that a repetition polls a position adjacent the position poled in the previous polling step for raw target sequencing reads having the consensus nucleobase polled in the previous step or in the case of a raw target sequencing read reassigned from the rejected class to the accepted class and not polled in the previous polling step or if polled not having the consensus nucleobase in the previous polling step, the polling polls a position adjacent the position aligned with the last nucleobase of the nascent sequence to determine a consensus nucleobase, and the consensus nucleobases determined in successive repetitions are assigned as successive nucleobases in the nascent sequence of the adjacent segment.
 2. The method of claim 1, wherein step (f) is performed at least once.
 3. The method of claim 1, wherein step (g) is performed at least 20 times and step (f) at least 5 times.
 4. The method of claim 1, wherein step (g) is performed at least 100 times and step (f) at least 20 times.
 5. The method of claim 1, wherein the threshold for step (f) is at least 80% identity between the raw target sequencing read and nascent sequence when maximally aligned and a match between the last assigned nucleobase of the nascent sequence and corresponding nucleobase of the raw targeting sequencing read.
 6. The method of claim 1, wherein the threshold level of accuracy of the sequencing the anchor segment is based on percentage of sequence identity and/or location of matched nucleobases between a raw target sequencing read and the known anchor segment.
 7. The method of claim 1, wherein the threshold level of accuracy requires a raw target sequencing read to have the correct nucleobase corresponding to the nucleobase of the anchor segment immediately adjacent the adjacent segment.
 8. The method of claim 1, wherein the nucleic acid target includes a nucleobase variation at a position and when step (g) polls the position it determines two consensus nucleobases for the position, wherein the nascent sequence is branched into two nascent sequences differing between the two consensus nucleobases and the consensus nucleobase determined in further repetitions of step (g) is assigned to both nascent sequences.
 9. The method of any of claim 1 wherein the nucleic acid target comprises first and second anchor segments at opposing ends of the nucleic acid target and the raw target sequencing reads include a first group of raw target sequencing reads of the first anchor segment and an adjacent segment and a second group of raw target sequencing reads of the second anchor segment and an adjacent segment; the first and second groups being raw sequencing reads of opposing strands of the nucleic acid target and the method is performed on the first and second groups of raw target sequencing reads to determine consensus sequences of opposing strands of the target nucleic acid.
 10. The method of claim 1, wherein the raw sequencing reads comprise raw sequencing reads of first and second nucleic acid targets, the first nucleic acid target comprising the anchor segment linked to a first adjacent segment and the second nucleic acid target comprising the anchor segment linked to a second adjacent segment.
 11. The method of claim 10, wherein the first and second adjacent segments are overlapping segments.
 12. The method of claim 11, wherein the first and second adjacent segments are fragments of the same contiguous polynucleotide.
 13. The method of claim 11, wherein the first and second adjacent segments are nonoverlapping segments.
 14. The method of claim 1, wherein the raw sequencing reads comprising raw sequence reads of a plurality of nucleic acid targets, the different nucleic acid targets comprising the anchor segment linked to different adjacent segments; the different adjacent segments including overlapping and nonoverlapping segments.
 15. The method of any of the preceding claims, wherein a strand of the anchor segment is a primer or primer binding site incorporated into the nucleic acid target.
 16. The method of any of the preceding claims, wherein a strand of the anchor segment has 4-120 nucleobases.
 17. The method of any of the preceding claims, wherein a strand of the anchor segment has 8-30 nucleobases.
 18. The method of claim 1, wherein the anchor segment is an oligonucleotide ligated to a nucleic acid fragment to be sequenced.
 19. The method of claim 1, wherein the anchor segment and adjacent segment are contiguous segments in a nucleic acid from nature.
 20. The method of claim 1, wherein the anchor segment is a repeat sequence.
 21. The method of any preceding claim further comprising outputting the sequence of at least part of the adjacent segment.
 22. The method of any preceding claim further comprising synthesizing a nucleic acid sequence having a sequence comprising at least part of the adjacent segment.
 23. The method of any of the preceding claims, further comprising experimentally determining the population of raw target sequencing reads of the target nucleic acid.
 24. The method of claim 23, wherein the population of raw target sequencing reads is determined by a sequencing-by-synthesis method.
 25. The method of claim 24, wherein the sequencing method is single-molecule sequencing.
 26. The method of claim 24, wherein the sequencing method is single-molecule real time sequencing.
 27. The method of claim 26, wherein the nucleic acid target is in the form of a circular template.
 28. The method of claim 1, wherein the nucleic acid target is a homogeneous population of the same nucleic acid molecule.
 29. The method of claim 1, wherein the nucleic acid target is a heterogeneous population of variant nucleic acid molecules.
 30. The method of claim 29, wherein the variant nucleic acid molecules are variant nucleic acid molecules of the same virus.
 31. The method of claim 30, wherein the virus is HIV or HCV.
 32. The method of claim 30, wherein the variants are allelic variants.
 33. The method of claim 1, wherein the nucleic acid target is a circular DNA molecule and the raw target sequencing reads comprise alternating reads of the anchor segment and the adjacent segment.
 34. The method of claim 33, wherein the reads of the adjacent segment comprise reads of alternating strands of the adjacent segment.
 35. The method of claim 33, wherein the circular DNA molecule is formed by ligating first and second hairpin anchor segments to the adjacent segment.
 36. The method of claim 35, wherein the first and second hairpin anchor segments are the same.
 37. The method of claim 35, wherein the first and second hairpin anchors are different and the raw target sequencing reads comprise alternating reads of the first and second hairpin anchor segments.
 38. The method of claim 1, further comprising, designating a segment of the nascent sequence of the adjacent segment as a new anchor segment and repeating the method to determine a consensus sequence of an adjacent segment adjacent the new anchor segment.
 39. A computer program product for analyzing a nucleic acid target, comprising (a) code for receiving a population of raw target sequencing reads of a nucleic acid target comprising an anchor segment and an adjacent segment; the anchor segment being of known sequence and the adjacent segment being of unknown sequence; and at least some of the raw target sequencing reads containing sequencing errors; (b) code for evaluating the accuracy of sequencing of the anchor segment in different raw target sequencing reads by comparing the raw target sequencing reads of the anchor segment with the known sequence of the anchor segment; (c) code for assigning a subset of the raw target sequencing reads into an accepted class based on reaching at least a threshold level of accuracy of the sequencing of the anchor segment; (d) code for polling nucleobases at a position equidistant to the anchor segment sequence in raw target sequencing reads in the accepted class to determine a consensus nucleobase, which consensus nucleobase is assigned as the first nucleobase of a nascent sequence of the adjacent segment; (e) code for assigning raw target sequencing reads having the consensus nucleobase determined in the prior polling step to remain in an accepted class and assigning raw target sequencing reads lacking the consensus nucleobase determined in the prior polling step to the rejected class; (f) code for optionally reassigning a raw target sequencing read from the rejected class to the accepted class by scoring similarity of the raw target sequencing read to the nascent sequence and reintroducing the raw target sequencing read if the sequence similarity reaches at least a threshold level of similarity; and (g) code for repeating steps coded in (d), (e) and optionally (f), except that a repetition polls a position adjacent the position poled in the previous polling step for raw target sequencing reads having the consensus nucleobase polled in the previous step or in the case of a raw target sequencing read reassigned from the rejected class to the accepted class and not polled in the previous polling step or if polled not having the consensus nucleobase in the previous polling step, the polling polls a position adjacent the position aligned with the last nucleobase of the nascent sequence to determine a consensus nucleobase, and the consensus nucleobases determined in successive repetitions are assigned as successive nucleobases in the nascent sequence of the adjacent segment.
 40. The computer program product of claim 39, wherein the threshold in (f) is at least 80% identity between the raw target sequencing read and nascent sequence when maximally aligned and a match between the last assigned nucleobase of the nascent sequence and corresponding nucleobase of the raw targeting sequencing read.
 41. The computer program product of claim 39, wherein the threshold level of accuracy of the sequencing the anchor segment is based on percentage of sequence identity and/or location of matched nucleobases between a raw target sequencing read and the known anchor segment.
 42. The computer program product of claim 39, wherein the threshold level of accuracy requires a raw target sequencing read to have the correct nucleobase corresponding to the nucleobase of the anchor segment immediately adjacent the adjacent segment.
 43. The computer program product of claim 39, wherein the raw sequencing reads comprise raw sequencing reads of first and second nucleic acid targets, the first nucleic acid target comprising the anchor segment linked to a first adjacent segment and the second nucleic acid target comprising the anchor segment linked to a second adjacent segment.
 44. The computer program product of claim 43, wherein the first and second adjacent segments are overlapping segments.
 45. The computer program product of claim 45, wherein the first and second adjacent segments are fragments of the same contiguous polynucleotide.
 46. The computer program product of claim 45, wherein the first and second adjacent segments are nonoverlapping segments.
 47. The computer program product of claim 39, wherein the raw sequencing reads comprise raw sequencing reads of a plurality of nucleic acid targets, the different nucleic acid targets comprising the anchor segment linked to different adjacent segments; the different adjacent segments including overlapping and nonoverlapping segments.
 48. The computer program product of any of claims 39-47, wherein the strand of the anchor segment is a primer incorporated into the nucleic acid target.
 49. The computer program product of any of claims 39-47, wherein the strand of the anchor segment has 4-120 nucleobases.
 50. The computer program product of any of claims 39-47, wherein the strand of the anchor segment has 8-30 nucleobases.
 51. The computer program product of claim 39, wherein the anchor segment is an oligonucleotide ligated to a nucleic acid fragment to be sequenced.
 52. The computer program product of claim 39, wherein the anchor segment and adjacent segment are contiguous segments in a nucleic acid from nature.
 53. The computer program product of claim 39, wherein the anchor segment is a repeat sequence.
 54. The computer program product of any of claims 39-53 further comprising code for outputting the sequence of at least part of the adjacent segment.
 55. The computer program product of claim 39, wherein the nucleic acid target is a homogeneous population of the same nucleic acid molecule.
 56. The computer program product of claim 39, wherein the nucleic acid target is a heterogeneous population of variant nucleic acid molecules.
 57. The computer program product of claim 56, wherein the variant nucleic acid molecules are variant nucleic acid molecules of the same virus.
 58. The computer program product of claim 57, wherein the virus is HIV or HCV.
 59. The computer program product of claim 57, wherein the variants are allelic variants.
 60. A system for analyzing a nucleic acid target, comprising: (1) a system bus; (2) a memory coupled to the system bus; and (3) a processor coupled to the system bus operatively disposed to: (a) receive a population of raw target sequencing reads of a nucleic acid target comprising an anchor segment and an adjacent segment; the anchor segment being of known sequence and the adjacent segment being of unknown sequence; and at least some of the raw target sequencing reads containing sequencing errors; (b) evaluate the accuracy of sequencing of the anchor segment in different raw target sequencing reads by comparing raw target sequencing reads of the anchor segment with the known sequence of the anchor segment; (c) assign a subset of the raw target sequencing reads into an accepted class based on reaching at least a threshold level of accuracy of the sequencing of the anchor segment; (d) poll nucleobases at a position equidistant to the anchor segment sequence in raw target sequencing reads in the accepted class to determine a consensus nucleobase, which consensus nucleobase is assigned as the first nucleobase of a nascent sequence of the adjacent segment; (e) assign raw target sequencing reads having the consensus nucleobase determined in the prior polling step to remain in an accepted class and assigning raw target sequencing reads lacking the consensus nucleobase determined in the prior polling step to the rejected class; (f) optionally reassign a raw target sequencing read from the rejected class to the accepted class by scoring similarity of the raw target sequencing read to the nascent sequence and reintroducing the raw target sequencing read if the sequence similarity reaches at least a threshold level of similarity; and (g) repeat steps (d), (e) and optionally (f), except that a repetition polls a position adjacent the position poled in the previous polling step for raw target sequencing reads having the consensus nucleobase polled in the previous step or in the case of a raw target sequencing read reassigned from the rejected class to the accepted class and not polled in the previous polling step or if polled not having the consensus nucleobase in the previous polling step, the polling polls a position adjacent the position aligned with the last nucleobase of the nascent sequence to determine a consensus nucleobase, and the consensus nucleobases determined in successive repetitions are assigned as successive nucleobases in the nascent sequence of the adjacent segment.
 61. The system of claim 60, wherein the threshold in (f) is at least 80% identity between the raw target sequencing read and nascent sequence when maximally aligned and a match between the last assigned nucleobase of the nascent sequence and corresponding nucleobase of the raw targeting sequencing read.
 62. The system of claim 60, wherein the threshold level of accuracy of sequencing the anchor segment is based on percentage of sequence identity and/or location of matched nucleobases between a raw target sequencing read and the known anchor segment.
 63. The system of claim 60, wherein the threshold level of accuracy requires a raw target sequencing read to have the correct nucleobase corresponding to the nucleobase of the anchor segment immediately adjacent the adjacent segment.
 64. The system of claim 60, wherein the raw sequencing reads comprise raw sequencing reads of first and second nucleic acid targets, the first nucleic acid target comprising the anchor segment linked to a first adjacent segment and the second nucleic acid target comprising the anchor segment linked to a second adjacent segment.
 65. The system of claim 60, wherein the first and second adjacent segments are overlapping segments.
 66. The system of claim 65, wherein the first and second adjacent segments are fragments of the same contiguous polynucleotide.
 67. The system of claim 65, wherein the first and second adjacent segments are nonoverlapping segments.
 68. The system of claim 62, wherein the raw sequencing reads comprising raw sequence reads of a plurality of nucleic acid targets, the different nucleic acid targets comprising the anchor segment linked to different adjacent segments; the different adjacent segments including overlapping and nonoverlapping segments.
 69. The system of any of claims 60-68, wherein the strand of the anchor segment is a primer incorporated into the nucleic acid target.
 70. The system of any of claims 60-68, wherein the strand of the anchor segment has 4-120 nucleobases.
 71. The system of any of claims 60-68, wherein the strand of the anchor segment has 8-30 nucleobases.
 72. The system of claim 60, wherein the anchor segment is an oligonucleotide ligated to a nucleic acid fragment to be sequenced.
 73. The system of claim 60, wherein the anchor segment and adjacent segment are contiguous segments in a nucleic acid from nature.
 74. The system of claim 60, wherein the anchor segment is a repeat sequence.
 75. The system of any of claims 60-74, wherein the processor is operatively disposed to outputting the sequence of at least part of the adjacent segment.
 76. The system of claim 60, wherein the nucleic acid target is a homogeneous population of the same nucleic acid molecule.
 77. The system of claim 60, wherein the nucleic acid target is a heterogeneous population of variant nucleic acid molecules.
 78. The system of claim 77, wherein the variant nucleic acid molecules are variant nucleic acid molecules of the same virus.
 79. The system of claim 78, wherein the virus is HIV or HCV.
 80. The system of claim 78, wherein the variants are allelic variants.
 81. A method of differentially treating a patient population, comprising; sequencing samples from members of the patient population; wherein for each sample the sequencing comprises (a) receiving a population of raw target sequencing reads of a nucleic acid target comprising an anchor segment and an adjacent segment; the anchor segment being of known sequence and the adjacent segment being of unknown sequence; and at least some of the raw target sequencing reads containing sequencing errors; (b) evaluating the accuracy of sequencing of the anchor segment in different raw target sequencing reads by comparing raw target sequencing read of the anchor segment with the known sequence of the anchor segment; (c) assigning a subset of the raw target sequencing reads into an accepted class based on reaching at least a threshold level of accuracy of the sequencing of the anchor segment; (d) polling nucleobases at a position adjacent the anchor segment sequence in raw target sequencing reads in the accepted class to determine a consensus nucleobase, which consensus nucleobase is assigned as the first nucleobase of a nascent sequence of the adjacent segment; (e) assigning raw target sequencing reads having the consensus nucleobase determined in the prior polling step to remain in an accepted class and assigning raw target sequencing reads lacking the consensus nucleobase determined in the prior polling step to the rejected class; (f) optionally reassigning a raw target sequencing read from the rejected class to the accepted class by scoring similarity of the raw target sequencing read to the nascent sequence and reintroducing the raw target sequencing read if the sequence similarity reaches at least a threshold level of similarity; and (g) repeating steps (d), (e) and optionally (f), except that a repetition polls a position adjacent the position poled in the previous polling step for raw target sequencing reads having the consensus nucleobase polled in the previous step or in the case of a raw target sequencing read reassigned from the rejected class to the accepted class and not polled in the previous polling step or if polled not having the consensus nucleobase in the previous polling step, the polling polls a position adjacent the position aligned with the last nucleobase of the nascent sequence to determine a consensus nucleobase, and the consensus nucleobases determined in successive repetitions are assigned as successive nucleobases in the nascent sequence of the adjacent segment; wherein different members of the patient population receive different treatment regimes depending on the determined sequence for the sample from each member.
 82. A computer-implemented method of analyzing a nucleic acid target, comprising (a) receiving a population of raw target sequencing reads of a nucleic acid target comprising an anchor segment and an adjacent segment; the anchor segment being of known sequence and the adjacent segment being of unknown sequence; and at least some of the raw target sequencing reads containing sequencing errors; (b) evaluating the accuracy of sequencing of the anchor segment in different raw target sequencing reads by comparing raw target sequencing reads of the anchor segment with the known sequence of the anchor segment; (c) assigning a subset of the raw target sequencing reads into an accepted class based on the accuracy of sequencing of the anchor segment in the raw target sequencing reads; and (d) determining a sequence of the anchor segment from raw target sequencing reads in the accepted class.
 83. The method of claim 82, wherein step (d) comprises polling nucleobases at corresponding positions in raw target sequencing reads in the accepted class to determine a consensus nucleobase, which consensus nucleobase is assigned as the first nucleobase of a nascent sequence of the adjacent segment and wherein step (d) is repeated and the consensus nucleobases determined in successive repetitions are assigned as successive nucleobases in the nascent sequence of the adjacent segment.
 84. The method of claim 83, further comprising assigning raw target sequencing reads having the consensus nucleobase determined in the prior polling step to remain in an accepted class and assigning raw target sequencing reads lacking the consensus nucleobase determined in the prior polling step to a rejected class.
 85. The method of claim 82, further comprising, designating a segment of the sequence of the adjacent segment as a new anchor segment and repeating the method to determine a sequence of an adjacent segment adjacent the new anchor segment.
 86. A computer program product for analyzing a nucleic acid target, comprising code for receiving a population of raw target sequencing reads of a nucleic acid target comprising an adapter segment and an adjacent segment; the adapter segment being of known correct sequence and the adjacent segment being of unknown sequence; and at least some of the raw target sequences containing sequencing errors; code for evaluating the accuracy of sequencing of the adapter segment in different raw target sequences by comparing raw target sequencing reads of the anchor segment with the known correct sequence of the adapter segment; code for assigning a subset of the raw target sequences into an accepted class based on the accuracy of sequencing of the adapter segment in the raw target sequences; code for aligning at least some of the raw target sequences from the accepted class; and code for determining a sequence of at least part of the adjacent segment from the aligned sequences.
 87. A system for analyzing a nucleic acid target, comprising: (a) a system bus; (b) a memory coupled to the system bus; and (c) a processor coupled to the system bus for receiving a population of raw target sequencing reads of a nucleic acid target comprising an adapter segment and an adjacent segment; the adapter segment being of known correct sequence and the adjacent segment being of unknown sequence; and at least some of the raw target sequences containing sequencing errors; evaluating the accuracy of sequencing of the adapter segment in different raw target sequences by comparing the raw target sequencing reads of the anchor segment with the known correct sequence of the adapter segment; assigning a subset of the raw target sequences into an accepted class based on the accuracy of sequencing of the adapter segment in the raw target sequences; aligning at least some of the raw target sequences from the accepted class; and determining a sequence of at least part of the adjacent segment from the aligned sequences.
 88. A method of differentially treating a patient population, comprising; sequencing samples from members of the patient population; wherein for each sample the sequencing comprises receiving a population of raw target sequencing reads of a nucleic acid target comprising an adapter segment and an adjacent segment; the adapter segment being of known correct sequence and the adjacent segment being of unknown sequence; and at least some of the raw target sequences containing sequencing errors; evaluating the accuracy of sequencing of the adapter segment in different raw target sequences by comparing raw target sequencing reads of the anchor segment with the known correct sequence of the adapter segment; assigning a subset of the raw target sequences into an accepted class based on the accuracy of sequencing of the adapter segment in the raw target sequences; aligning at least some of the raw target sequences from the accepted class; and determining a sequence of at least part of the adjacent segment from the aligned sequences; wherein different members of the patient population receive different treatment regimes depending on the determined sequence for the sample from each member. 